Nucleic acid cloning

ABSTRACT

The present invention provides an improved system for linking nucleic acids to one another. In particular, the present invention provides techniques for producing DNA product molecules that may be easily and directly ligated to recipient molecules. The product molecules need not be cleaved with restriction enzymes in order to undergo such ligation. In preferred embodiments of the invention, the DNA product molecules are produced through iterative DNA synthesis reactions, so that the product molecules are amplified products. The invention further provides methods for directed ligation of product molecules (i.e., for selective ligation of certain molecules within a collection of molecules), and also for methods of exon shuffling, in which multiple different product molecules are produced in a single ligation reaction. Preferred embodiments of the invention involve ligation of product molecules encoding functional protein domains, particularly domains naturally found in conserved gene families. The inventive DNA manipulation system is readily integrated with other nucleic acid manipulation systems, such as ribozyme-mediated systems, and also is susceptible to automation.

The present application is a Continuation of co-pending U.S. Nationalpatent application Ser. No. 09/897,712, filed Jun. 29, 2001, which is aU.S. National Application corresponding to International Application No.PCT/US00/00189 filed Jan. 5, 2000 under 35 USC § 271, and which isfurther a Continuation-in-part of co-pending U.S. National patentapplication Ser. No. 09/225,990, filed Jan. 5, 1999. This applicationalso claims priority to U.S. Provisional patent application Ser. No.60/114,909, filed on Jan. 5, 1999. Each of these priority applicationsis incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

Some or all of the work described herein was supported by grant numberRO1 GM 52409 from the National Institutes of Health and by grant numberMCB9604458 from the National Science Foundation; the United StatesGovernment may have certain rights in this invention.

BACKGROUND

The Molecular Biology revolution began with the discovery of enzymesthat were capable of cleaving double stranded DNA, so that DNA fragmentswere produced that could be ligated to one another to generate new,so-called “recombinant” molecules (see, for example, Cohen et al., Proc.Natl. Acad. Sci. USA 70:1293, 1973; Cohen et al., Proc. Natl. Acad. Sci.USA 70:3274, 1973; see also U.S. Pat. Nos. 4,740,470; 4,468,464;4,237,224). The revolution was extended by the discovery of thepolymerase chain reaction (PCR), which allowed rapid amplification ofparticular DNA segments, producing large amounts of material that couldsubsequently be cleaved and ligated to other DNA molecules (see, forexample, U.S. Pat. Nos. 4,683,195; 4,683,202; 5,333,675).

Despite the power of these digestion and amplification techniques,however, there remains substantial room for improvement. Reliance ondigesting enzymes, called “restriction enzymes”, can render molecularbiological experiments quite expensive. Moreover, many of the enzymesare inefficient or are only available in crude preparations that may becontaminated with undesirable entities.

At first, it seemed that PCR amplification might itself avoid many ofthe difficulties associated with traditional cut-and-paste cloningmethods since it was thought that PCR would generate DNA molecules thatcould be directly ligated to other molecules, without first beingcleaved with a restriction enzyme. However, experience indicates thatmost PCR products are refractory to direct cloning. One possibleexplanation for this observation has come from research revealing thatmany thermophilic DNA polymerases (including Taq, the most commonly usedenzyme) add terminal 3′-dAMP residues to the products they amplify.Invitrogen (Carlsbad, Calif.) has recently developed a system for directcloning of such terminally-dAMP-tagged products (TA Cloning Kit®; seeU.S. Pat. No. 5,487,993) if the molecule to which they are to be ligatedis processed to contain a single unpaired 3′-dTMP residue. While theInvitrogen system has proven to be very useful, it is itself limited inapplication by being restricted to ligation of products with only asingle nucleotide overhang (an A residue), and is further restricted inthat the overhang must be present at the 3′ end of the DNA molecule tobe ligated.

There is a need for the development of improved systems for nucleic acidcloning. Particularly desirable systems would allow DNA ligation withminimal reliance on restriction enzymes, would provide for efficientligation, and would be generally useful for the ligation of DNAs havinga wide variety of chemical structures. Optimal systems would evenprovide for directional ligation (i.e., ligation in which the DNAmolecules to be linked together will only connect to one another in oneorientation).

SUMMARY OF THE INVENTION

The present invention provides an improved system for linking nucleicacids to one another. In particular, the present invention providestechniques for producing DNA product molecules that may be easily anddirectly ligated to recipient molecules. The product molecules need notbe cleaved with restriction enzymes in order to undergo such ligation.In preferred embodiments of the invention, the DNA product molecules areproduced through iterative DNA synthesis reactions, so that the productmolecules are amplified products.

The inventive system provides techniques and reagents for generatingproduct molecules with 3′ overhangs, 5′ overhangs, or no overhangs, andfurther provides tools for ligating those product molecules withrecipient molecules. Where overhangs are employed, the length andsequence of the overhang may be varied according to the desires of thepractitioner. Overhang-containing products may be linked to one anotherby any available means including, for example, enzymatic ligation ortransformation into a host cell. For example, molecules containing atleast 12 nt overhangs may be annealed to one another and linked togetherby transformation into E. coli without first being ligated (see, forExample, Rashtchian, et al. Annalytical Biochemistry 206:91, 1992).

The inventive system further provides methods for directed ligation ofproduct molecules (i.e., for selective ligation of certain moleculeswithin a collection of molecules), and also for methods of exonshuffling, in which multiple different product molecules are produced ina single ligation reaction. Preferred embodiments of the inventioninvolve ligation of product molecules encoding functional proteindomains, particularly domains naturally found in conserved genefamilies. Alternative or additional preferred embodiments of theinvention involve multi-component ligation reactions, in which three ormore nucleic acid molecules are ligated together. In some embodiments,these multiple molecules are linked in only a single arrangement; inothers, multiple arrangements can be achieved.

The inventive DNA manipulation system is readily integrated with othernucleic acid manipulation systems, such as ribozyme-mediated systems,and also is susceptible to automation. Specifically, in one aspect, adouble stranded DNA molecule with a single stranded overhang comprisedof RNA is provided. Additionally, in another aspect, a library ofnucleic acid molecules is provided wherein each member of the librarycomprises 1) at least one nucleic acid portion that is common to allmembers of the library; and 2) at least two nucleic acid portions thatdiffer in different members of the library, is also provided by thepresent invention. In a preferred embodiment, each of the nucleic acidportions in the library comprises protein-coding sequence and eachlibrary member encodes a continuous polypeptide. In yet anotherparticularly preferred embodiment, each of the variable nucleic acidportions encodes a functional domain of a protein. This functionaldomain is preferably one that is naturally found in a gene familyselected from the group consisting of the tissue plasminogen activatorgene family, the animal fatty acid synthase gene family, the polyketidesynthase gene family, the peptide synthetase gene family, and theterpene synthase gene family.

In yet another aspect of the present invention, a method of generating ahybrid double-stranded DNA molecule is provided. This method comprisesthe steps of 1) providing a first double-stranded DNA molecule, whichdouble-stranded DNA molecule contains at least one single strandedoverhang comprised of RNA; 2) providing a second double-stranded DNAmolecule containing at least one single-strand overhang that iscomplementary to the RNA overhang on the first double-stranded DNAmolecule; and 3) ligating the first and second double-stranded DNAmolecules to one another so that a hybrid double-stranded DNA moleculeis produced. In certain preferred embodiments, the method comprisesproviding and ligating at least three double-stranded DNA molecules.

A further aspect of the present invention includes a method ofgenerating a hybrid double-stranded DNA molecule, the methodcomprising 1) generating a first double-stranded DNA molecule byextension of first and second primers, at least one of which includes atleast one base that is not copied during the extension reaction so thatthe extension reaction produces a product molecule containing a firstoverhang; 2) providing a second double-stranded DNA molecule containinga second overhang complementary to the first overhang; and 3) ligatingthe first and second double-stranded DNA molecules to one another, sothat a hybrid double-stranded DNA molecule is produced. In certainpreferred embodiments, the method comprises providing and ligating atleast three double-stranded DNA molecules.

In still a further aspect of the present invention, a method ofgenerating a hybrid double-stranded DNA molecule is provided, the methodcomprising: 1) generating a first double-stranded DNA molecule byextension of first and second primers, at least one of which includes atleast one potential point of cleavage; 2) exposing the firstdouble-stranded DNA molecule to conditions that result in cleavage ofthe cleavable primer at the potential point of cleavage, so that a firstoverhang is generated on the first DNA molecule; 3) providing a seconddouble-stranded DNA molecule containing a second overhang complementaryto the first overhang; and 4) ligating the first and seconddouble-stranded DNA molecules to one another, so that a hybriddouble-stranded DNA molecule is produced. In certain preferredembodiments, the method comprises providing and ligating at least threedouble-stranded DNA molecules.

DESCRIPTION OF THE DRAWING

FIG. 1 depicts an inventive process for generating DNA product moleculeswith 3′ overhangs.

FIG. 2 depicts a process for producing 5′ overhangs by hybridizing atemplate molecule with one or more primers including at least oneribonucleotide primer.

FIG. 3 depicts an inventive process for generating DNA product moleculeswith one or more 5′ overhangs.

FIG. 4 depicts an alternative inventive process for generating DNAproduct molecules with one (FIG. 4A) or more (FIG. 4B) 5′ overhangs.

FIG. 5 presents a process that allows ligation of blunt-ended molecules.

FIG. 6 shows members of the tissue plasminogen activator gene family.

FIG. 7 presents a list of certain polyketide compounds that arecurrently used as pharmaceutical drugs for the treatment of human andanimal disorders.

FIG. 8 depicts the different functional domains of bacterial polyketidesynthase genes responsible for the production of erythromycin andrapamycin.

FIG. 9 depicts the different functional domains of bacterial polyketidesynthase genes responsible for the production of erythromycin andrapamycin.

FIG. 10 depicts the protein functional domains of certain modularpolyketide synthase genes.

FIG. 11 presents a list of products generated by peptide synthetasesthat are currently used as pharmacologic agents.

FIG. 12 depicts the protein functional domains of certain modularpeptide synthetase genes.

FIG. 13 depicts the structure of the srfA peptide synthetase operon.

FIG. 14 depicts the synthesis of isoprenoids through the polymerizationof isoprene building blocks.

FIG. 15 depicts certain cyclization and intermolecular bond formationreactions catalyzed by isoprenoid, or terpene synthases.

FIG. 16 presents a schematic illustration of the correspondence betweennatural exons and functional domains within isoprenoid synthases.

FIG. 17 depicts one generic example of a directional ligation reaction.

FIG. 18 presents a schematic representation of an inventive specificdirectional ligation reaction.

FIG. 19A depicts the nucleotide sequence of the glutamate receptor exonsknown as Flip (GenBank accession number X64829).

FIG. 19B depicts the nucleotide sequence of the glutamate receptor exonsutilized are known as Flop (GenBank accession number X64830).

FIG. 20 shows the amplified hybrid molecules produced in an inventivedirectional ligation reaction.

FIG. 21 presents the nucleotide sequence of the ligation junction in thehybrid molecules of FIG. 20.

FIG. 22 presents the nucleotide sequence of the human β-globin gene.

FIG. 23 shows an inventive identity exon shuffling reaction.

FIG. 24 shows an inventive positional exon shuffling reaction.

FIG. 25 shows the combinatorial potential of certain inventive directedligation techniques.

FIG. 26 presents one version of a combinedprimer-based/ribozyme-mediated nucleic acid manipulation schemeaccording to the present invention.

FIG. 27 depicts a robotic system that could be utilized in the practiceof certain inventive methods.

FIG. 28 depicts a schematic representation of a directional ligationreaction employing inventive product molecules containing 3′ overhangs.

FIG. 29 presents a schematic of certain bioassay techniques that can beemployed to determine the success of primer copying and/or ligation ininventive reactions.

FIG. 30 shows a ribozyme mediated directional ligation reaction.

FIG. 31 shows constructs employed in the reaction of FIG. 30.

FIGS. 32 and 33 show products of the reaction of FIG. 30.

FIG. 34 shows a variety of chimeras generated using DNA-Overhang Cloning(“DOC”). The parental genes qare shown in lines 1 and 2. The fivechimeric genes are shown below the parental genes. Jagged edges indicatethat only a portion of introns 13 and 15 were amplified. Lengths ofchimeric genes (in basepairs) are indicated.

DEFINITIONS

“Cloning”—The term “cloning”, when used herein, means the production ofa new nucleic acid molecule through the ligation of previously unlinkednucleic acid pieces to one another. A molecule produced by such ligationis considered a “clone” for the purposes of the present application,even before it has been replicated.

“Direct ligation”—The term “direct ligation”, as applied to productmolecules herein, means that a product molecule may be ligated to one ormore recipient molecules without first being cleaved with a restrictionenzyme. Preferably, no processing of the product molecule is required atall prior to ligation.

“Expression”—“Expression” of nucleic acid sequences, as that term isused herein, means that one or more of (i) production of an RNA templatefrom a DNA sequence; (ii) processing (e.g., splicing and/or 3′ endformation) of a pre-mRNA to produce an mRNA; and (iii) translation of anmRNA has occurred.

“Gene”—For the purposes of the present invention, the term “gene” hasits art understood meaning. However, it will be appreciated by those ofordinary skill in the art that the term “gene” has a variety of meaningsin the art, some of which include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences, and others of whichare limited to coding sequences. It will further be appreciated that artdefinitions of “gene” include references to nucleic acids that do notencode proteins but rather encode functional RNA molecules, such astRNAs. For the purpose clarity, we note that, as used in the presentapplication, the term “gene” generally refers to a portion of a nucleicacid that encodes a protein; the term may optionally encompassregulatory sequences. This definition is not intended to excludeapplication of the term “gene” to non-protein-coding expression units,but rather to clarify that, in most cases, the term as used in thisdocument happens to be applied to a protein-coding nucleic acid.

“Gene fragment”—A “gene fragment”, as that term is used herein, means apiece of a protein-coding DNA molecule that is shorter than the completeprotein-coding molecule. Preferably, the fragment is at least about 12bases long, more preferably at least about 15-20 bases long, and may beseveral hundred or thousands of base pairs long. It should be understoodthat the fragment need not include protein-coding sequence, but rathermay represent a non-coding portion of the original gene.

“Hybrid nucleic acid”—A “hybrid nucleic acid”, as that term is usedherein, means a nucleic acid molecule comprising at least a firstsegment and a second segment, each of which occurs in nature but is notlinked directly with the other in nature, the first and second segmentsbeing directly linked to one another in the hybrid nucleic acid.

“Overhang sequence”—An “overhang sequence”, as that term is used herein,means a single stranded region of nucleic acid extending from a doublestranded region.

“Primer”—The term “primer”, as used herein, refers to a polynucleotidemolecule that is characterized by an ability to be extended against atemplate nucleic acid molecule, so that a polynucleotide molecule whosesequence is complementary to that of at least a portion of the templatemolecule, is linked to the primer. Preferred primers are at leastapproximately 15 nt long. Particularly preferred primers have a lengthwithin the range of about 18-30, preferably longer than approximately 20nucleotides

“Product molecule”—A “product molecule”, as that term is used herein, isa nucleic acid molecule produced as described herein. Preferably, theproduct molecule is produced by extension of an oligonucleotide primeraccording to the present invention. A product molecule may be singlestranded or double stranded. In certain preferred embodiments of theinvention, a product molecule that includes a double-stranded portionalso includes a single-stranded 3′- or 5′-overhang. In other preferredembodiments, the product molecule is blunt-ended. Where a productmolecule is produced in an iterative DNA synthesis reaction (e.g., a PCRreaction), it is referred to as an “amplified product”.

“Recipient molecule”—A “recipient molecule”, as that term is usedherein, is a nucleic acid molecule to which a product molecule is to beligated. The recipient molecule may be, but is not required to be, avector. In general, the recipient molecule can be any molecule selectedby the practitioner.

“Vector”—A “vector”, as that term is used herein, is a nucleic acidmolecule that includes sequences sufficient to direct in vivo or invitro replication of the molecule. Where the vector includes in vivoreplication sequences, these sequences may be self-replicationsequences, or sequences sufficient to direct integration of the vectorinto another nucleic acid already present in the cell, so that thevector sequences are replicated during replication of thealready-present nucleic acid. Such already-present nucleic acid may beendogenous to the cell, or may have been introduced into the cellthrough experimental manipulation. Preferred vectors include a cloningsite, at which foreign nucleic acid molecules, preferably inventiveproduct molecules, may be introduced and ligated to the vectors.Particularly preferred vectors further include control sequencesselected for their ability to direct in vivo or in vitro expression ofnucleic acid sequences introduced into the vector. Such controlsequences may include, for example, transcriptional control sequences(e.g., one or more promoters, regulator binding sites, enhancers,terminators, etc.), splicing control sequences (e.g., one or more splicedonor sites, splice acceptor sites, splicing enhancers, etc.), andtranslational control sequences (e.g., a Shine Dalgarno sequence, astart codon, a termination codon, etc.). Vectors may also include somecoding sequence, so that transcription and translation of sequencesintroduced into the vector results in production of a fusion protein.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

Product Molecules with 3′ Overhangs

In one aspect, the present invention provides reagents and methods forgenerating product molecules with 3′ overhangs that can be directlyligated to recipient molecules. The length and sequence of the 3′overhang may be determined by the user.

FIG. 1 depicts one embodiment of this aspect of the invention. As shownin that Figure, first and second primers are provided that flank atarget region of a template nucleic acid molecule. At least one of theprimers includes one or more ribonucleotides at its 5′ end.Specifically, if primer 1 is x nucleotides long and primer 2 is ynucleotides long, then n1=a whole number (including 0) from 0 to x andn2=a whole number (including 0) from 0 to y except that (i) n1 and n2cannot both be 0; and (ii) n1 can only be x (or n2 can only be y) if theDNA polymerase employed in the extension reaction is capable ofextending an RNA primer. The characteristics (e.g., ability to extend anRNA primer, ability to copy RNA into DNA [whether the RNA is presentedalone or as part of a hybrid RNA/DNA molecule) of a wide variety of DNApolymerases are well known in the art (see, for example, manufacturer'scatalogs, Myers et al., Biochem. 6:7661, 1991), and where suchcharacteristics are not known for a particular DNA polymerase, routineassays are available for determining them (see, for example, Bebenek etal., Met. Enzymol. 262:217, 1995; see also Example 3).

In certain preferred embodiments of the invention, each of primers 1 and2 includes at least one 5′-terminal ribonucleotide residue. In otherpreferred embodiments, at least one primer includes at least 2ribonucleotide residues, one of which is the 5′-terminal residue. Theprimer may include at least 3, 4, 5, 6-10, or more ribonucleotideresidues and even, as mentioned above, may be entirely RNA. Preferably,the ribonucleotide residues are contiguous with one another.

The nucleotide sequence of each of primer 1 and primer 2 is selected bythe practitioner and need not be fully complementary with the sequenceof the target nucleic acid. As is known in the art, perfectcomplementarity is not required for successful DNA synthesis, though itis generally desired that at least the 3′-terminal nucleotide of theprimer be perfectly paired with the template. The 5′ end of the primer,however, need not be paired at all, and it is common in the art to addadditional sequences to a target sequence by including them in theprimer. Of course, it is also acceptable for the primer to include aportion, 5′ of the extendible 3′ terminus, that does not hybridize withthe template, and also to include a yet more 5′ portion that doeshybridize with the template. For the purposes of the present invention,any such variation on primer sequence, or any other available variation,is acceptable, so long as (i) at least one primer includes aribonucleotide that either is present at the 5′ end of the primer orwill generate a new 5′ end of the primer upon being removed from theprimer (e.g., by alkaline treatment, preferably followed by kinasetreatment); and (ii) each primer hybridizes sufficiently well andsufficiently specifically under the conditions of the reaction that aproduct molecule is produced.

Other considerations of primer design are well known in the art (see,for example, Newton et al. (eds), PCR: Essential Data Series, John Wiley& Sons, New York, N.Y., 1995; Dieffenbach (ed), PCR Primer: a LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1995; White et al. (eds), PCR Protocols: Current Methods andApplications; Methods in Molecular Biology, The Humana Press, Totowa,N.J., 1993; Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego, Calif., 1990; Griffin et al.(eds), PCR Technology, Current Innovations, CRC Press, Boca Raton, Fla.1994, each of which is incorporated herein by reference). For instance,it is often desirable for approximately 50% of the hybridizing residuesto be Gs or Cs; and may be desirable, for optimal extension, for the3′-terminal residue to also be a G or a C.

Primers such as those depicted in FIG. 1, that contain at least oneribonucleotide residue as their 5′ terminal residue (or as a residuewhose removal will create a new 5′-terminal primer residue), may beprepared by any technique available in the art. For example, suchprimers may be chemically synthesized. Companies (e.g., Oligos, Etc.,Inc., Bethel, Me.) that supply oligonucleotide reagents will typicallyprepare hybrid RNA/DNA oligonucleotides, or RNA only nucleotides, aspreferred by the practitioner. Alternatively, RNA sequences may beligated to DNA sequences using standard techniques (see, for example,Moore et al., Science 256:992, 1992; Smith (ed), RNA: ProteinInteractions, A Practical Approach, Oxford University Press, 1998, whichparticularly discusses construction of RNA molecules containingsite-specific modifications by RNA ligation; each of these references isincorporated herein by reference).

As shown in FIG. 1, an extension reaction is performed so that DNAsynthesis is primed from each of the first and second primers, and adouble stranded DNA/RNA hybrid molecule is created with at least oneribonucleotide residue at the 5′ end of at least one strand. Preferably,but not essentially, the DNA polymerase utilized in the extensionreaction is one that does not add extraneous 3′ nucleotides. Also, asmentioned above, if one or both of the primers has a ribonucleotide asits 3′ residue, the DNA polymerase utilized in the extension step mustbe one that is capable of extending from a ribonucleotide primer.

FIG. 1 shows that the hybrid molecule is then exposed to a treatmentthat removes the ribonucleotide residues. As depicted in FIG. 1, thattreatment is exposure to elevated pH (e.g., treatment with a base suchas sodium hydroxide [NaOH]). Any other treatment that removes RNAresidues without disturbing DNA residues (e.g., exposure to RNase, etc.)could alternatively be employed at this step.

When the ribonucleotide residues are removed from the hybrid molecule,the resultant molecule is left with a double stranded portion and asingle stranded 3′ overhang on at least one of its ends. FIG. 1 depictsa product molecule with single-stranded 3′ overhangs at both ends. Thesequence and length of the overhang was determined by the sequence andlength of RNA present at the 5′ end of the primers. Clearly, anysequence and length of overhang can be selected. In certain preferredembodiments of the invention, the sequence and length of the overhangcorresponds with that produced by cleavage of double-stranded DNA by acommercially available restriction enzyme, so that the product moleculecan be ligated to recipient molecules that have been cut with thatenzyme. A variety of enzymes that leave 3′ overhangs are known in theart, including but not limited to AatII, AlwnI, NsiI, SphI, etc.

In other preferred embodiments, the 3′ overhang sequence and length isselected to base pair with a 3′ overhang generated in another inventiveproduct molecule, so that the two molecules may readily be ligatedtogether (see, for example, Example 1).

Furthermore, it will be appreciated that the 3′ overhangs at the twoends of the product molecule need not have the same sequence or length(see, for example, Example 1). It is often desirable to generate anucleic acid molecule that can be ligated to a recipient molecule inonly one orientation, or that can be ligated to two different recipientnucleic acid molecules (e.g., a three-way ligation) in a particulararrangement. Accordingly, it is quite valuable to be able to engineerthe sequence and length of the 3′ overhangs of the inventive productmolecule.

As can be seen with reference to FIG. 1, the nature of the ends left bythe ribonucleotide-removal treatment can affect the behavior of theproduct molecule in subsequent ligation reactions. In particular,alkaline hydrolysis of ribonucleotides leaves 5′-OH groups rather than5′-phosphate groups. As is known in the art, at least one terminalphosphate group is typically required for successful ligation of nucleicacid molecules. Thus, if the product molecule depicted in FIG. 1 is tobe ligated to a recipient molecule that lacks the appropriate terminalphosphate groups (e.g., because of exposure to treatment with aphosphatase), it will be desirable to add 5′ phosphate groups to therecipient molecule prior to ligation. Any available technique may beutilized to achieve such phosphate group addition; most commonly, thephosphate groups will be added by treatment with polynucleotide kinase.

The product molecules depicted in FIG. 1 may be ligated to any desiredrecipient molecule. Preferably, the recipient molecule has at least one3′ overhang that is complementary to at least a portion of the at leastone 3′ overhang on the product molecule. It will be appreciated that, ifthe recipient molecule has a 3′ overhang whose 3′ terminal portion iscomplementary to the 3′ terminal portion of the product molecule 3′overhang, but is not otherwise complementary to the product molecule 3′overhang, then one or more gaps will be present after hybridization,which gaps can be filled in with DNA polymerase prior to ligation. Sincethe sequence and length of the product molecule 3′ overhang is selectedby the practitioner, this approach may be employed to add sequence tothe recombinant molecule that would not be present if complete 3′overhang hybridization had occurred. For the purposes of the presentinvention, the complementary 3′-terminal portions of the product andrecipient molecules should be at least one nucleotide long, and can be2, 3, 4, 5, 6-10 nucleotides long, or longer. In certain preferredembodiments, the complementary 3′-terminal portions are less than about6 nucleotides long, so that efficiency of ligation (usually performed at4° C. or 14° C.) is preserved and complications associated withannealing longer sequences are avoided.

Preferred recipient molecules include, but are not limited to,linearized vectors. Such vectors may be linearized, for example bydigestion with a restriction enzyme that produces a 3′ overhangcomplementary to that present on the product molecule. Alternatively,such linearized vectors may be prepared as product molecules asdescribed herein, containing one or more 3′ overhangs selected by thepractitioner to be compatible with the 3′ overhangs present on otherproduct molecules.

Those of ordinary skill in the art will appreciate that productmolecules can readily be generated according to the present invention sothat each end of a given product molecule has a different 3′ overhang.Such molecules can be used in directional cloning experiments, wherethey can be ligated to one or more other molecules in only a singleorientation. Such directional ligation strategies are particularlyuseful where three or more molecules are desired to be linked to oneanother. In such multi-component ligation reactions, it is often usefulto minimize the possibility of self-ligation by individual molecules,and also to reduce the chance that the molecules will link together withone or more molecule being in an improper orientation.

Product Molecules with 5′ Overhangs

FIGS. 2-4 depict inventive strategies for producing product moleculeswith 5′ overhangs. For example, as shown in FIG. 2, a template moleculemay be hybridized with one or more primers including at least oneribonucleotide. For this embodiment of the present invention, it is notrequired that the ribonucleotide be located at the 5′ end of theoligonucleotide, though such is acceptable. The primer may contain 2, 3,4, 5, 6-10, or more ribonucleotides, and may be wholly ribonucleotidesif the DNA polymerase utilized in the extension reaction will extend aribonucleotide primer. That is, in FIG. 2, at least one of n1 and n2 isa whole number greater than or equal to 1, and n3 and n4 are each awhole number greater than or equal to zero. The particular inventiveembodiment depicted in FIG. 3 utilizes two primers. Those of ordinaryskill in the art will appreciate that each primer includes a portion,terminating with the 3′-terminal residue of the primer, that hybridizessufficiently well with the template molecule to allow extension. Thesequence of the remainder of the primer, however, need not becomplementary to that of the template molecule. Furthermore, those ofordinary skill in the art will also appreciate that if the DNApolymerase being employed includes a 3′→5′ exonuclease activity, it isnot even essential that the 3′-most residue in the primer hybridize withthe template, so long as the exonuclease activity is allowed to chewback to a point in the primer from which extension can occur.

After hybridization with the primer(s), an extension reaction isperformed with a DNA polymerase that does not copy ribonucleotides. Forexample, we have found that Vent_(R) and Vent_(R)® (exo⁻) do not useribonucleotide bases as a template (see Example 1); Tth and Taqpolymerases, by contrast, are reported to be able to replicateribonucleotides (Myers et al., Biochem. 6:7661, 1991), as, of course,are reverse transcriptases. Other DNA polymerases may be tested fortheir ability to copy ribonucleotides according to standard techniquesor, for example, as described in Example 3.

The extension reaction shown in FIG. 2 may be iterated as anamplification reaction, if desired. The embodiment depicted in FIG. 2illustrates such an amplification, from which the product is a doublestranded molecule with two 5′ overhangs, each of which includes at leastone ribonucleotide residue. Those of ordinary skill in the art willappreciate that the sequence and length of each 5′ overhang (as well asis ribonucleotide composition) is selected by the practitioner, and thatthe two product molecule overhangs depicted may be the same ordifferent.

This product molecule may then be hybridized with one or more recipientmolecules containing a 5′ overhang that is complementary to at least the5′-terminal residue of the product molecule. If gaps remain afterhybridization, they may be filled in with DNA polymerase according toknown techniques. If the gaps encompass a ribonucleotide residue, it maybe preferable to employ a DNA polymerase that will copy RNA in order toensure that the gap is filled. As mentioned above, such DNA polymerasesinclude, for example, Tth, Taq, and reverse transcriptase. Other DNApolymerases may be tested for their ability to copy RNA according toknown techniques or, for example, as described in Example 3.

Once any gaps are filled, the product and recipient molecules may beligated together. DNA ligase is known to close nicks (i) betweenadjacent deoxyribonucleotides; (ii) between a deoxyribonucleotide and aribonucleotide; or (iii) between adjacent ribonucleotides. Thus, ahybrid molecule can be produced containing both DNA and RNA residues.This molecule can be copied into DNA, either in vitro according tostandard techniques, or in vivo after introduction in to a host cellcapable of copying such a molecule (Escherichia coli, for example, havebeen reported to be able to remove and replace ribonucleotides that arebase-paired with deoxyribonucleotides—see Sancar, Science 266:1954,1994). Alternatively, it may be desirable to replicate the hybridizedcompound into DNA rather than performing a ligation (e.g., by PCR withDNA primers or with a DNA polymerase that can copy ribonucleotides).Also, it should be mentioned that, in some cases, ligation may beaccomplished in vivo rather than in vitro, as is known in the art forexample for co-transformation of yeast cells.

As depicted in FIG. 2, the product molecule is ligated with only asingle recipient molecule and at only one end. Those of ordinary skillin the art will appreciate that a product molecule may alternatively beligated at both of its ends, either to a single recipient molecule or totwo different recipient molecules.

FIG. 3 presents an alternative approach to generating product moleculeswith one or more 5′ overhangs. In this embodiment, instead of employingribonucleotide primer residues and a DNA polymerase that cannot copyRNA, we utilize a modified base in the primer, which modified base isnot copied by the DNA polymerase. A wide variety of modified nucleotidesare known in the art (see, for example, U.S. Pat. No. 5,660,985; seealso various catalogs such as that provided by Oligos, Etc. [Bethel,Me.]); those that are not copied by particular DNA polymerases may beidentified, for example, by reference to the manufacturer's catalog, byroutine screening according to known techniques, or as described, forexample, in Example 3.

Modified bases may be removed from the product molecule, before or afterits ligation to a recipient molecule, either by DNA replication in vitroor in vivo with a DNA polymerase that will copy the modified base or byremoval of the base followed by gap repair, according to standardtechniques (see, for example, Sancar, Science 266:1954, 1994).

FIG. 4 presents an inventive embodiment for generating a productmolecule with at least one 5′ overhang. In the particular embodimentdepicted in FIG. 4, the inventive strategy is applied to a startingmolecule containing one (FIG. 4A) or two (FIG. 4B) 3′ overhangs, so thatthe starting molecule is converted from a 3′-overhang-containingcompound to a 5′-overhang-containing molecule. However, those ofordinary skill in the art will appreciate that the same approach couldequally well be applied to add one or two 5′ overhangs to a startingmolecule that is either blunt ended, or contains one or two 3′ or 5′overhangs.

The starting molecule depicted in FIG. 4 may be obtained by anyavailable means. The molecule may have one or two 3′ overhangs (meaningthat at least one of R1 and R2 is at least one nucleotide long) and maybe produced, for example, by restriction endonuclease cleavage of aprecursor fragment, by polymerase chain amplification, or by any othermeans. In certain preferred embodiments of the invention, the startingmolecule is produced by PCR and contains a single 3′ dATP at each end,as described above. FIG. 4A depicts the application of the inventivemethod to a starting molecule having only one 3′ overhang; FIG. 4Bdepicts the application of the inventive method to a starting moleculehaving two 3′ overhangs.

With reference to FIG. 4A, the starting molecule is hybridized with atleast one primer containing a first portion that hybridizes with a firstsequence in the starting molecule that is substantially adjacent to thestarting molecule 3′ overhang residue, a second portion that aligns withand fails to hybridize to at least one residue of the starting molecule3′ overhang, and a third portion that does not align with the startingmolecule but rather extends past (5′ in the primer) the last residue ofthe starting molecule 3′ overhang.

The length and sequence of the first portion of the primer is determinedby the sequence of the starting molecule adjacent the starting molecule3′ overhang. Hybridization by the first portion of the primer may extendinto the 3′ overhang, so long as at least one residue of the 3′ overhangis aligned with and fails to hybridize with the second portion of theprimer. The length and sequence of the second portion of the primer isdetermined to some degree by the length and sequence of the startingmolecule 3′ overhang in that the second portion must fail to hybridizewith at least one residue of the 3′ overhang, preferably but notessentially at least the 3′-terminal residue. So long as suchhybridization is avoided, the precise sequence of this second portion ofthe primer may be selected by the practitioner. The length (i.e., thevalue of n in FIG. 4A, which must be a whole number greater than orequal to 1) and sequence (i.e., the identities of N in FIG. 4A) of thethird portion of the primer is also determined by the practitioner. Thisthird portion will become a 5′ overhang in the product molecule.

As depicted in FIG. 4A, single or multiple rounds of extension from theinventive primer is performed. It will be appreciated by those ofordinary skill in the art that, due to the absence of a second primer(and the mismatch between the primer and the starting molecule 3′overhang, which prevents extension of the 3′ end of that strand of thestarting molecule) only linear, and not exponential, extension isaccomplished. Of course, if the DNA polymerase employed in the extensionreaction is one that adds one or more terminal 3′ residues, the productmolecule may have a 3′ overhang as well as a 5′ overhang.

Once the product molecule with a 5′ overhang is produced, it may behybridized with any recipient molecule that also contains a 5′ overhang,at least part of which is complementary to part of the product molecule5′ overhang. The hybridized compound contains a nick on each strand (ormay even contain a gap if the 5′ overhangs of the product and recipientmolecules are imperfectly matched in length) and at least one mismatchimmediately prior to the product molecule 5′ overhang. This hybridizedcompound is then exposed to a 3′→5′ exonuclease activity to remove themismatched base(s) (that correspond to the portion of the startingmolecule 3′ overhang that did not hybridize with the second portion ofthe primer). The digested compound is then exposed to a DNA polymeraseto fill in the gap created by exonuclease digestion, and subsequently toligase to heal any remaining nicks. Enzymes having 3′→5′ exonucleaseactivity are well known in the art (including, for example, E. coli DNApolymerase I, Pfu, Vent_(R)®, Deep Vent_(R)®, etc.); other enzymes maybe tested for this ability according to standard techniques.

Those of ordinary skill in the art will appreciate that the methoddepicted in FIG. 4A may be applied to either strand of a startingmolecule, depending on where the 3′ overhang is located. As depicted inFIG. 4B, the method may even be applied to both strands simultaneously,although it is important for such an embodiment to perform only a singleround extension reaction or to perform independent extension reactionsfor each strand. Amplification (i.e., multiple rounds of denaturationand extension) is not performed because such amplification would resultin the production of a blunt-ended molecule (or one with 3′ overhangs ifa DNA polymerase that adds 3′ nucleotides were employed), having thesequence dictated by the primers, rather than a molecule with a 5′overhang and a mismatch immediately 3′ of the 5′ overhang.

As shown in FIG. 4B, a starting molecule containing two 3′ overhangs isconverted to a product molecule containing two 5′ overhangs byapplication of the inventive method. The starting molecule is hybridizedwith two inventive primers containing first, second, and third portionsas described above in the discussion of FIG. 4A. Each primer is thenextended in single-round (or independent) extension reactions. It willbe understood by those of ordinary skill in the art that both extensionreactions need not be performed simultaneously, or on the same exactstarting molecule. Extensions of each primer can even be performed indifferent reaction vesicles.

Each of the double-stranded molecules produced in the extension reactionhas a single 5′ overhang, whose sequence and length corresponds to thatof the third primer portion. The strands of these double strandedmolecules are then separated from one another. Individual strands may beseparately purified if desired, but such is not required. Strands arethen mixed together (if they are not already together) and annealed, sothat the two new strands synthesized by extension of the primers havethe opportunity to anneal to one another. The product of this annealingreaction is an inventive product molecule with two 5′ overhangs. As willbe appreciated, these overhangs may be the same or different in lengthand/or sequence.

This product molecule may be hybridized with one or more recipientmolecules, each of which has a 5′ overhang whose 5′-terminal portion (atleast one nucleotide in length) is complementary with a 5′-terminalportion (of the same length) of the product molecule 5′ overhang. Anygaps remaining after hybridization may be filled in with a DNApolymerase; the product and recipient molecules may then be ligatedtogether.

Blunt-Ended Product Molecules

FIG. 5 presents an inventive embodiment that allows ligation ofblunt-ended molecules. As shown, blunt ended starting molecules areprovided that are to be linked together. Such molecules may be preparedby any available technique including, for example, digestion of aprecursor with one or more restriction enzymes (optionally followed by afill-in or chew-back of any overhanging ends), PCR (e.g., with a DNApolymerase that does not add extraneous 3′ nucleotides—reference can bemade to manufacturer's catalogs to determine the characteristics of aparticular DNA polymerase. For example, Vent_(R)® is reported togenerate >95% blunt ends; Vent_(R)® (exo⁻) is reported to generate about70% blunt ends and 30% single nucleotide 3′ overhangs, of anynucleotide; Pfu is reported to produce only blunt-ended molecules),chemical synthesis, etc. The starting molecules may be double strandedor single stranded. As depicted in FIG. 5, the starting molecules aredouble stranded.

The starting molecules are hybridized to bridging molecules, each ofwhich hybridizes to at least one terminal residue of two differentstarting molecules that are to be linked together. Clearly, if thestarting molecules are double stranded, they should be denatured priorto exposure to the bridging molecules, so that successful hybridizationwith the bridging molecules may occur. The bridging molecules mayhybridize to more than one residue of each starting molecule, and/or maycontain non-hybridizing portions between the portions that hybridize tothe two starting molecules. Also, the bridging molecules may havesufficient length that they abut one another after hybridization, or maybe short enough that gaps are present in the hybridized compound betweenthe individual bridging molecules. Preferably, at least one primerhybridizes to the 3′-terminus of the 3′-most starting molecule in thehybridized compound. This primer may extend past the terminus ifdesired, so that a 5′ overhang is created. No such overhang is depictedin FIG. 5.

The hybridized compound is then converted into a double-stranded DNAmolecule by any collection of available techniques. For example, gapsmay be filled with DNA polymerase and any remaining nicks sealed withDNA ligase. Or, if no gaps are present in one strand, that strand mayfirst be ligated and DNA polymerase subsequently applied, in vitro or invivo to seal gaps in the other strand or to synthesize a replacementstrand (e.g., primed from the bridging molecule hybridized at the most3′ location with respect to the starting molecules). In one preferredembodiment of the invention, gaps are filled and nicks sealed and theentire recombinant molecule is then replicated by PCR amplification. Ifdesired, a DNA polymerase that adds one or more 3′-terminal residues maybe employed, so that the resultant amplified product is likely to haveone or more 3′ overhangs. As described above, such a product may readilybe ligated to another molecule with complementary 3′ overhangs, such asoccurs in the use of the Invitrogen TA Cloning Kit® system.

Applications

The product molecules and ligation strategies provided above are usefulin any of a variety of contexts. For the purposes of clarification only,and not for limitation, we discuss certain of these contexts in moredetail here.

As described above, the present invention produced techniques andreagents for providing nucleic acid molecules that can be directlyligated (i.e., without first being digested with a restriction enzyme)to other molecules. The invention also provides techniques foraccomplishing such ligation. The present invention may be used to linknucleic acid molecules of any sequence to one another and therefore hasthe broadest possible application in the field of genetic cloning.

Those of ordinary skill in the art will appreciate that the inventivetechniques and reagents may be employed to link any DNA molecule to anyother DNA molecule, regardless of the particular sequences of the DNAmolecules, their protein-coding capacities, or any othercharacteristics. This feature distinguishes the present system fromtraditional, restriction-endonuclease-reliant cloning systems, for whichthe precise sequences of the molecules being linked can often affect thedesign of the cloning strategy, as it may be desirable, for example, toavoid cleaving one fragment with a particular enzyme that produces anundesired cleavage in another fragment, or to make other adjustments toaccommodate the behavior of the protein enzymes being employed.

Production of Protein-Coding Genes

In certain preferred embodiments of the present invention, one or moreof the DNA molecules included in an inventive ligation reaction includesopen reading frame, i.e., a protein-coding sequence. In particularlypreferred embodiments, at least two DNA molecules to be ligated togetherinclude open reading frame sequences, and their ligation produces ahybrid DNA containing both open reading frames linked together so that asingle polypeptide is encoded. Where ligation of two or more DNAmolecules, according to the present invention, generates at least oneopen reading frame that spans at least one ligation junction, theligation is considered to have generated a new, hybrid protein-codinggene.

In but one embodiment of the inventive system used to produceprotein-coding genes, the DNA molecules to be ligated to one another areselected to encode one or more discrete functional domains of knownbiological activity, so that the ligation of two or more such DNAmolecules produces a hybrid gene encoding a bi- or multi-functionalpolypeptide. It is well known in the art that many proteins havediscrete functional domains (see, for example, Traut, Mol. Cell.Biochem. 70:3, 1986; Go et al., Adv. Biophys. 19:91, 1985). It is alsowell known that such domains may often be separated from one another andligated with other discrete functional domains in a manner thatpreserves the activity of each individual functional domain.

Those of ordinary skill in the art will appreciate that some flexibilityis allowed in the selection of precise DNA sequences encoding functionalprotein domains. For example, it is often not desirable to limit the DNAsequences to only those that encode for exactly the amino acid residuescontained in a functional domain of a naturally-occurring protein.Additional DNA sequences may be included, for example, encoding linkersequences that can provide flexibility between the particular selectedfunctional domain and any other functional domain to which it is to belinked.

Alternatively or additionally, in some contexts researchers have foundthat it is useful to select DNA sequences encoding less than all of theamino acids comprising a particular functional domain (see, for example,WO 98/01546); in such cases, the other amino acids can be added back asa result of the subsequent ligation (i.e., can be encoded by anadjacently-ligated DNA molecule), or can be left out completely. Thoseof ordinary skill in the art will readily be able to familiarizethemselves with the application of these basic principles to theirparticular experimental question after appropriate consultation with theliterature describing the protein domains in which they are interested.

To give but a few examples of the types of functional protein domainsthat could be encoded by individual DNA molecules, or combinations ofDNA molecules, to be ligated according to the present invention, wellknown modular domains include, for example DNA binding domains (such aszinc fingers, homeodomains, helix-turn-helix motifs, etc.), ATP or GTPbinding domains, transmembrane spanning domains, protein-proteininteraction domains (such as leucine sippers, TPR repeats, WD repeats,STYX domains [see, for example, Wishart et al., Trends Biochem. Sci.23:301, 1998], etc.), G-protein domains, tyrosine kinase domains (see,for example, Shokat, Chem. Biol. 2:509, 1995), SRC homology domains(see, for example, Sudol, Oncogene 17:1469, 1998), SH2 domains (see, forexample, Schaffhausen, Biochim. Biophys. Acta 28:61, 1995), PTB domains(see, for example, van der Greer et al., Trends Biochem Sci 20:277,2995), the PH domain (see, for example, Musacchio et al., Trends BiochemScie 18:343, 1993), certain catalytic domains, cell surface receptordomains (see, for example, Campbell et al., Immunol. Rev. 163:11, 1998),carbohydrate recognition domains (see, for example, Kishore et al.,Matrix Biol. 15:583, 1997), immunoglobulin domains (see, for example,Rapley, Mol. Biotechnol. 3:139, 1995), etc. (see also, Hegyi et al., J.Protein. Chem. 16:545, 1997; Baron et al., Trends Biochem. Sci. 16:13,1997).

Typically, such domains are identified by homology comparisons thatidentify regions of sequence similarity within proteins of knownbiological activity (at least as relates to the portion of the proteinshowing the homology). The spatial coherence of any particularfunctional domain is often confirmed by structural studies such as X-raycrystallography, NMR, etc.

According to the present invention, a useful “functional domain” of aprotein is any portion of that protein that has a known biologicalactivity that is preserved with the portion is separated from the restof the protein, even if the portion must continue to be embedded withina larger polypeptide molecule in order to maintain its activity. Therelevant biological activity need not, and typically will not,constitute the complete biological activity of a particular protein inwhich the domain is naturally found, but rather will usually representonly a portion of that activity (e.g., will represent an ability to bindto a particular other molecule but will not include a further activityto cleave or modify the bound molecule). As noted, many such domainshave already been described in the literature; others can be identifiedby homology search, preferably in combination with mutational studies asis known in the art to define sequences that participate in biologicalactivity.

The present invention encompasses the recognition, now virtuallyuniversally accepted, that the production of new genes during evolutionhas often involved the novel combination of DNA sequences encoding twoor more already-existing functional protein domains (see, for example,Gilbert et al., Proc Natl Acad Sci USA, 94:7698, 1997; Strelets, et al.,Biosystems, 36:37, 1995). In fact, protein “families” are often definedby their common employment of particular functional domains, even thoughthe overall biological roles played by different family members may bequite unrelated (see further discussion of such families below, insection discussing exon shuffling). The present invention thereforeprovides techniques and reagents that can be used to mimic anevolutionary process in the laboratory. The universality andexperimental simplicity of the system provide researchers, who mayselect particular DNA modules to link to one another in desired orders,with significant advantages over Mother Nature, who must wait forstochastic processes to produce interesting new results.

Accordingly, preferred protein functional domains to be employed inaccordance with the present invention include those that have beenre-used through evolution to generate gene families (i.e., collectionsof genes that encode different members of protein families). Exemplarygene families created by re-use of particular protein domains include,for example, the tissue plasminogen activator gene family (see, forexample FIG. 6); the family of voltage-gated sodium channels (see, forexample, Marban et al., J. Physiol. 508:647, 1998); certain families ofadhesion molecules (see, for example, Taylor et al., Curr. Top.Microbiol. Imunol. 228:135, 1998); various extracellular domain proteinfamilies (see, for example, Engel, Matrix Biol. 15:295, 1996; Bork, FEBSLett. 307:49, 1992; Engel, Curr. Opin. Cell. Biol. 3:779, 1991); theprotein kinase C family (see, for example, Dekker et al., Curr. Op.Struct. Biol. 5:396, 1995); the tumor necrosis factor receptorsuperfamily (see, for example, Naismith et al., J. Inflamm 47:1, 1995);the lysin family (see, for example, Lopez et al., Microb. Drug Resist.3:199, 1997); the nuclear hormone receptor gene superfamily (see, forexample, Ribeiro et al., Annu. Rev. Med. 46:443, 1995; Carson-Jurica etal., Endocr. Rev. 11:201, 1990); the neurexin family (see, for example,Missler et al., J. Neurochem. 71:1339, 1998); the thioredoxin genefamily (see, for example, Sahrawy et al., J. Mol. Evol., 42:422, 1996);the phosphoryl transfer protein family (see, for example, Reizer et al.,Curr. Op. Struct. Biol. 7:407, 1997); the cell wall hydrolase family(see, for example, Hazlewood et al., Prog. Nuc. Acid Res. Mol. Biol.61:211, 1998); as well as certain families of synthetic proteins (e.g.,fatty acid synthases, polyketide synthases [see, for example, WO98/01546; U.S. Pat. No. 5,252,474; U.S. Pat. No. 5,098,837; EP PatentApplication Number 791,655; EP Patent Application Number 791,656],peptide synthetases [see, for example, Mootz et al., Curr. Op. Chem.Biol. 1:543, 1997; Stachelhaus et al., FEMS Microbiol. Lett 125:3,1995], and terpene synthases).

The present invention allows DNA molecules encoding different functionaldomains present in these families to be linked to one another togenerate in-frame fusions, so that hybrid genes are produced that encodepolypeptides containing different arrangements of the selectedfunctional domains. It will be appreciated that experiments can beperformed in which (i) only the domains utilized in a particular genefamily in nature are linked to one another (in new arrangements), or inwhich (ii) domains naturally utilized in different gene families arelinked to one another.

In one particularly preferred embodiment of the present invention, theDNA modules selected to be ligated together comprise modules encoding atleast one functional domain, or portion of a functional domain, of amember of a synthetic enzyme family. As mentioned above, a variety ofenzyme families are known whose members are responsible for thesynthesis of related biologically active compounds. Families ofparticular interest include the fatty acid synthase family, thepolyketide synthase family, the peptide synthetase family, and theterpene synthase family (sometimes called the terpenoid synthase family,or the isoprenoid synthase family). The individual members of theseenzyme families are multi-domain proteins that catalyze the synthesis ofparticular biologically active chemical compounds. For any particularfamily member, different protein domains catalyze different steps in theoverall synthesis reaction. Each family member catalyzes the synthesisof a different chemical compound because each contains a differentcollection or arrangement of protein functional domains. As will beunderstood in the context of the present application, the instantinvention provides a system by which the various protein domainsutilized in these gene families may be linked to one another in newways, to generate novel synthase enzymes that will catalyze theproduction of new chemical entities expected to have biologicalactivities related to those produced by naturally-occurring members ofthe gene family from which the functional domains were selected.

In order to more clearly exemplify this aspect of the present invention,we discuss below certain characteristics and attributes of each of theabove-mentioned particularly preferred synthetic enzyme proteinfamilies:

Animal Fatty Acid Synthase Family

The aminal fatty acid synthase comprises two multifunctional polypeptidechains, each of which contains seven discrete functional domains. Fattyacid molecules are synthesized at the interface between the twopolypeptide chains, in a reaction that involves the iterativecondensation of an acetyl moiety with successive malonyl moieties (see,for example, Smith, FASEB J. 8:1248, 1994; Wakil, Biochemistry 28:4523,1989, each of which is incorporated herein by reference). Most commonly,the β-keto intermediate produced in this condensation reaction iscompletely reduced to produce palmitic acid; in certain instances,however, alternative substrates or alternative chain-terminatingmechanisms are employed so that a range of products, includingbranched-chain, odd carbon-numbered, and shorter-chain-length fatty acidmolecules. These molecules have a range of roles in biological systems,including (i) acting as precursors in the production of a variety ofsingalling molecules, such as steroids, as well as (ii) participating inthe regulation of cholesterol metabolism.

Those of ordinary skill in the art, considering the present disclosure,will readily recognize that the techniques and reagents described hereincan desirably be applied to DNA molecules encoding one or more of thefunctional domains of a fatty acid synthase molecule, so that themolecules may be linked to other DNA molecules to create interesting newhybrid DNAs, preferably encoding hybrid animal fatty acid synthase genesthat may have novel synthetic capabilities.

Polyketide Synthase Family

Polyketides represent a large and structurally diverse class of naturalproducts that includes many important antibiotic, antifungal,anticancer, antihelminthic, and immunosuppressant compounds such aserythromycins, tetracylcines, amphotericins, daunorubicins, avermectins,and rapamycins. For example, FIG. 7 presents a list of certainpolyketide compounds that are currently used as pharmaceutical drugs inthe treatment of human and animal disorders.

Polyketides are synthesized by protein enzymes, aptly named polyketidesynthases, that catalyze the repeated stepwise condensation ofacylthioesters in a manner somewhat analogous to that employed by thefatty acid synthases. Structural diversity among polyketides isgenerated both through the selection of particular “starter” or“extender” units (usually acetate or proprionate units) employed in thecondensation reactions, and through differing degrees of processing ofthe β-keto groups observed after condensation. For example, some β-ketogroups are reduced to β-hydroxyacyl-groups; others are both reduced tothis point, and are subsequently dehydrated to 2-enoyl groups; stillothers are reduced all the way to the saturated acylthioester.

Polyketide synthases (PKSs) are modular proteins in which differentfunctional domains catalyze different steps of the synthesis reactions(see, for example, Cortes et al., Nature 348:176, 1990; MacNeil et al.,Gene 115:119, 1992; Schwecke et al., Proc. Natl. Acad. Sci. USA 92:7839,1995). For example, FIGS. 8 and 9 (from WO 98/01546) depict thedifferent functional domains of bacterial polyketide synthase genesresponsible for the production of erythromycin and rapamycin,respectively (see also FIG. 10). Each of these genes is an example of aso-called “class I” bacterial PKS gene. As shown, each cycle ofpolyketide chain extension is accomplished by a catalytic unitcomprising a collection of functional domains including a β-ketoacyl ACPsynthase domain (KS) at one end and an acyl carrier protein (ACP) domainat the other end, with one or more other functional domains (selectedfrom the group consisting of an acyl transferase [AT] domain, aβ-ketoacyl reductase [KR] domain, an enoyl reductase [ER] domain, adehydratase [DH] domain, and a thioesterase [TE] domain).

Class II bacterial PKS genes are also modular, but encode only a singleset of functional domains responsible for catalyzing chain extension toproduce aromatic polyketides; these domains are re-used as appropriatein successive extension cycles (see, for example, Bibb et al., EMBO J.8:2727, 1989; Sherman et al., EMBO J. 8:2717, 1989; Femandez-Moreno etal., J. Biol. Chem. 267:19278, 1992; Hutchinson et al., Annu. Rev.Microbiol. 49:201, 1995). Diversity is generated primarily by theselection of particular extension units (usually acetate units) and thepresence of specific cyclases (encoded by different genes) that catalyzethe cyclization of the completed chain into an aromatic product.

It is known that various alterations in and substitutions of class I PKSfunctional domains can alter the chemical composition of the polyketideproduct produced by the synthetic enzyme (see, for example, Cortes etal., Science 268:1487, 1995; Kao et al., J. Am. Chem. Soc. 117:9105,1995; Donadio et al., Science 252:675, 1991; WO 93/1363). For class IIPKSs, it is known that introduction of a PKS gene from one microbialstrain into a different microbial strain, in the context of a differentclass II PKS gene cluster (e.g., different cyclases) can result in theproduction of novel polyketide compounds (see, for example, Bartel etal., J. Bacteriol. 172:4816, 1990; WO 95/08548).

The present invention provides a new system for generating altered PKSgenes in which the arrangement and/or number of functional domainsencoded by the altered gene differs from that found in anynaturally-occurring PKS gene. Any PKS gene fragment can be used inaccordance with the present invention. Preferably, the fragment encodesa PKS functional domain that can be linked to at least one other PKSfunctional domain to generate a novel PKS enzyme. A variety of differentpolyketide synthase genes have been cloned (see, for example, Schweckeet al., Proc. Natl. Acad. Sci. USA 92:7839, 1995; U.S. Pat. No.5,252,474; U.S. Pat. No. 5,098,837; EP Patent Application Number791,655; EP Patent Application Number 791,656, each of which isincorporated herein by reference; see also WO 98/51695, WO 98/49315, andreferences cited therein, also incorporated by reference.), primarilyfrom bacterial or fungal organisms that are prodigious producers ofpolyketides. Fragments of any such genes may be utilized in the practiceof the present invention.

Peptide Synthetase Family

Peptide synthetases are complexes of polypeptide enzymes that catalyzethe non-ribosomal production of a variety of peptides (see, for example,Kleinkauf et al., Annu. Rev. Microbiol. 41:259, 1987; see also U.S. Pat.No. 5,652,116; U.S. Pat. No. 5,795,738). These complexes include one ormore activation domains (DDA) that recognize specific amino acids andare responsible for catalyzing addition of the amino acid to thepolypeptide chain. DDA that catalyze the addition of D-amino acids alsohave the ability to catalyze the recemization of L-amino acids toD-amino acids. The complexes also include a conserved thioesterasedomain that terminates the growing amino acid chain and releases theproduct. FIG. 11 presents an exemplary list of products generated bypeptide synthetases that are currently being used as pharmacologicagents.

The genes that encode peptide synthetases have a modular structure thatparallels the funcitonal domain structure of the enzymes (see, forexample, Cosmina et al., Mol. Microbiol. 8:821, 1993; Kratzxchmar etal., J. Bacteriol. 171:5422, 1989; Weckermann et al., Nuc. Acids res.16:11841, 1988; Smith et al., EMBO J. 9:741, 1990; Smith et al., EMBO J.9:2743, 1990; MacCabe et al., J. Biol. Chem. 266:12646, 1991; Coque etal., Mol. Microbiol. 5:1125, 1991; Diez et al., J. Biol. Chem.265:16358, 1990; see also FIG. 12). For example, FIG. 13 (from U.S. Pat.No. 5,652,116) presents the structure of one exemplary peptidesynthetase gene operon, the srfA operon.

The sequence of the peptide produced by a particular peptide synthetaseis determined by the collection of functional domains present in thesynthetase. The present invention, by providing a system that allowsready linkage of particular peptide synthetase functional domains to oneanother, therefore provides a mechanism by which new peptide synthasegenes can be produced, in which the arrangement and/or number offunctional domains is varied as compared with naturally-occurringpeptide synthase genes. The peptide synthase enzymes encoded by such newgenes are expected to produce new peptide products. The presentinvention therefore provides a system for the production of novelpeptides, through the action of hybrid peptide synthase genes.

Terpene Synthase Family

Isoprenoids are chemical compounds whose structure represents amodification of an isoprene building block. The isoprenoid familyincludes a wide range of structurally diverse compounds that can bedivided into classes of primary (e.g., sterols, carotenoids, growthregulators, and the polyprebol substitutents of dolichols, quinones, andproteins) and secondary (e.g., monoterpenes, sesquiterpenes, andditerpenes) metabolites. The primary metabolites are important forbiological phenomena such as the preservation of membrane integrity,photoprotection, orchestration of developmental programs, and anchoringof essential biochemical activities to specific membrane systems; thesecondary metabolites participate in processes involving inter-cellularcommunication, and appear to mediate interactions between plants andtheir environment (see, for example, Stevens, in Isopentoids in Plants[Nes et al., eds], Macel Dekker et al., New York, pp. 65-80, 1984;Gibson et al., Nature 302:608, 1983; and Stoessl et al., Phytochemistry15:855, 1976).

Isoprenoids are synthesized through the polymerization of isoprenebuilding blocks, combined with cyclization (or other intramolecular bondformation) within intermediate or final product molecules. Thepolymerization reactions are catalyzed by prenyltransferases that directthe attack of an electron deficient carbon on the electron-rich carbonatom in the double bond on the isoprene unit (see FIG. 14, from U.S.Pat. No. 5,824,774). Cyclizations and other intramolecular bondformation reactions are catalyzed by isoprenoid, or terpene, synthases(see FIG. 15, from U.S. Pat. No. 5,824,774).

The terpene synthase proteins are modular proteins in which functionaldomains tend to correspond with natural exons (see, for example, U.S.Pat. No. 5,824,774, incorporated herein by reference). FIG. 16, fromU.S. Pat. No. 5,824,774, presents a schematic illustration of thecorrespondence between natural exons and funcitonal domains withinisoprenoid synthases. The upper diagram represents the organization ofexons within the TEAS gene, which is nearly identical to that of the HVSand casbene synthase genes; the lower diagram shows the alignment offunctional domains to the exonic organization of the TEAS and HVS genes.

As will be appreciated in light of the present application, the instantinvention provides a system by which DNA molecules encoding isoprenoidsynthase functional domains may be linked to one another to generatenovel hybrid isoprenoid synthase genes in which the arrangement and/ornumber of functional domains is varied as compared with those observedin naturally-occurring isoprenoid synthase genes. These novel hybridgenes will encode novel hybrid proteins that are expected to catalyzethe synthesis of new isoprenoid compounds.

As mentioned above, in some embodiments of the invention, DNA moleculesencoding functional domains from one protein family are linked to DNAmolecules encoding functional domains from a different protein family.Of particular interest in accordance with the present invention arereactions in which DNAs encoding polyketide synthase functional domainsare linked with DNAs encoding peptide synthetase functional domains.Alternative preferred embodiments involve linkage of fatty acid synthasefunctional domains with either or both of polyketide synthase functionaldomains and peptide synthetase functional domains. The hybrid genescreated by such inter-family ligation reactions can then be testedaccording to known techniques to determine their ability to encodeproteins that catalyze the synthesis of novel chemical compounds relatedto polyketides, fatty acids, and/or peptides.

As also mentioned above, it will be appreciated that the DNA moleculesselected to be linked to one another in a particular experiment are notlimited to molecules encoding functional domains or portions thereof;molecules encoding “linker” amino acids may additionally oralternatively be employed, as can non-coding molecules, depending on thedesired final product.

To give but one example, it may sometimes be desirable to include in afinal ligated molecule certain control sequences that will regulateexpression of other DNA sequences to which the control sequences arelinked when the ligated molecule is introduced into a host cell or an invitro expression system. For example, transcriptional control sequences,RNA splicing control sequences, other RNA modification controlsequences, and/or translational control sequences may be included in oneor more of the DNA molecules to be linked together. A wide variety ofsuch expression control sequences are well known in the art (see, forexample, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989,incorporated herein by reference); those of ordinary skill in the artwill be familiar with considerations relevant to selecting desirablecontrol sequences for use in their particular application. In general,so long as such control sequences direct expression of other DNAsequences to which they are linked when those DNA sequences areintroduced into a cell or an in vitro expression system, they areappropriate for use in accordance with the present invention.

Other DNA modules that could desirably be used in accordance with thepresent invention include, for example, modules encoding a detectableprotein moiety (e.g., an enzyme moiety that catalyzes a detectablereaction such as a color change or induction of fluorescence orluminescence, or a moiety that interacts with a known monoclonalantibody, etc), modules encoding a moiety that allows ready purificationof any polypeptide encoded by the ligated product DNA molecule (e.g., aGST domain, a copper chelate domain, etc.), or any other module desiredby the researcher.

Directional Ligation

As discussed herein, one particularly valuable application of theinventive techniques is for the linkage of multiple different nucleicacid molecules to one another. Because the embodiments of the inventionthat provide product molecules with 3′ or 5′ overhangs allow thesequence and length of those overhangs to be selected at thepractitioner's discretion, molecules can readily be prepared forligation only to certain designated partners, in certain designatedorders, so that multi-member ligation reactions can be performed withonly minimal generation of spurious or undesired ligation products.

FIG. 17 presents a schematic depiction of one generic example of such adirectional ligation reaction according to the present invention (FIGS.18-22 and Example 1 describe a specific example). As shown, a firstnucleic acid molecule, designated “A”, contains a first overhang,designated “overhang 1” on one end. A second nucleic acid molecule, “B”is flanked by a second overhang, “overhang 1′”, that is complementary tooverhang 1, and a third overhang, “overhang 2”, that is preferablyunrelated to, and certainly not identical with, overhang 1, A thirdnucleic acid molecule, “C”, contains a fourth overhang, “overhang 2′”,that is complementary with overhang 2. As will be appreciated by thoseof ordinary skill in the art, a ligation reaction including all three ofthese nucleic acid molecules will produce only a single reactionproduct, “ABC”, and will not produce “AC” or circular “B” products dueto the incompatibility of the ends that would have to be ligatedtogether to generate such products.

Mutagenesis

In another particularly useful application, the inventive techniques andreagents may be utilized to alter the nucleotide sequence of nucleicacid molecules that are being linked together. A separate mutagenesisreaction is not required. Rather, primers and/or overhangs whosesequence and length may be selected by the practitioner are utilized tocreate single-stranded regions between molecules to be ligated, whichsingle-stranded regions include new or altered sequences as desired.These single-stranded regions can subsequently be filled in with apolymerase that will synthesize a strand complementary to the newsequence. Alternatively or additionally, primers may be employed thatadd sequence to a particular product molecule strand that will be copiedin an extension or amplification reaction.

Exon Shuffling

One particular application of the techniques and reagents describedherein is in the production of libraries of hybrid nucleic acidmolecules in which particular collections of DNA molecules, or “exons”have been linked to one another. That is, an “exon shuffling” reactionis one in which a single reaction mixture (e.g., a ligation mixture or asplicing reaction—discussed further below) generates at least two, andpreferably at least 10, 100, 1000, 10,000, 100,000, or 1,000,000different product molecules.

As used herein, the term “exon” refers to any DNA molecule that is to beligated to another DNA molecule. An exon may include protein-codingsequence, may be exclusively protein-coding, or may not includeprotein-coding sequence at all. The term “exon shuffling” is intended toindicate that, using the techniques and reagents of the presentinvention, collections of exons can be produced that can be ligated toone another in more than one possible arrangement. For example, asdepicted in FIG. 23, the inventive techniques and reagents may beemployed in a ligation reaction in which a single upstream exon, A, canbe ligated to any one of a collection of different internal exons(B1′-B4 in FIG. 23), which in turn is further ligated to a downstreamexon, C.

Those of ordinary skill in the art will readily appreciate that FIG. 23presents just one particular embodiment of an “identity exon shuffling”reaction (i.e., one in which the identity of a particular exon isdifferent in different products of the shuffling reaction) according tothe present invention. A wide array of related reactions is includedwithin the inventive “exon shuffling” concept, and particularly withinthe concept of “identity exon shuffling”. For example, more than oneexon may be varied in a particular shuffling reaction. In fact, it isnot necessary to have upstream and downstream terminal exons that areuniform among shuffling products, as is depicted in FIG. 23. Suchconsistency may provide certain advantages, however, including anability to amplify all shuffling products with a single set ofamplification primers (discussed in more detail below). Even ifinvariant flanking exons are preserved, however, more than one internalexon may be varied; even if additional invariant internal exons are alsoprovided.

FIG. 24 presents an embodiment of a different sort of exon shufflingreaction that may be performed according to the present invention. Inthe particular embodiment shown in FIG. 24, upstream (A) and downstream(H) exons are provided in combination with a wide variety of possibleinternal exons (B-G). All exons have compatible overhangs. In such areaction, the possibilities for internal exons arrangements to be foundin product molecules are infinite. Also, because no exons (other thanthe optional flanking exons) are restricted to a particular position inthe exon chain, this type of shuffling is referred to as “positionalshuffling”.

Of course, those of ordinary skill in the art will appreciate that FIG.24 is but an exemplary embodiment of inventive positional shufflingsystems. For example, it may well be desirable to employ at least twosets of compatible overhangs and to ensure that potential internal exonsare not flanked by compatible ends; otherwise, intramolecularcircularization can present serious complications as a competingreaction in inventive ligations. Also, it is possible to perform an exonshuffling reaction that represents a compromise between the extremes ofallowing identity shuffling at a single position while holding all otherpositions fixed (e.g., FIG. 17) and allowing complete shuffling at allpositions. Merely by selecting the compatibility of the overhangs, thepractitioner may limit the number of exons able to incorporate at aparticular chain site, while allowing more variability at a differentsite.

One of the advantages of the present invention is that it allowssimultaneous multi-site variation, optionally in combination withpositional variation (i.e., the possibility that a particular exonsequence could end up in different positions in different productmolecules. To give but one example of the significance of thisphenomenon, FIG. 25 shows that other techniques might allow productionof libraries in which a single position in an exon chain can be variedat one time. For a three-exon chain in which 10 different exons could beemployed at each of the positions, 30 different variants can be produced(A1BC, A2BC, A3BC, . . . A10BC, AB1C, AB2C, . . . AB10C, ABC1, ABC2, . .. ABC10). By contrast, if all three positions can be variedsimultaneously, as is possible in accordance with the present invention,1000 different variants can be produced.

As discussed above, it is now accepted that the evolutionary processoften produces new genes by re-sorting existing exons. Large genefamilies have apparently been produced by exon shuffling. According tothe present invention, it is desirable to employ the inventivetechniques both to link particular selected functional domains to oneanother (see above) and to shuffle exons found in those gene families,so that a library of (at least two) product genes is generated.

The inventive exon shuffling techniques may be applied to any desiredcollection of exons. Preferably, they are applied to exons includingprotein-coding sequences. More preferably, they are applied toprotein-coding exons that have been re-used in evolution in differentmembers of gene families (see discussion above). In one particularlypreferred embodiment of the exon shuffling system of the presentinvention, the exons to be shuffled represent functional domains ofsynthetic enzymes. As discussed above with respect to ligation, re-sortexons from within family or between or among families.

Particularly preferred gene families to which inventive exon shufflingtechniques may be applied include, but are not limited to, the tissueplasminogen activator gene family, the animal fatty acid synthase genefamily, the polyketide synthase gene family, the peptide synthetase genefamily, and the terpene synthase gene family. The class I bacterialpolyketide synthase gene family presents a particularly attractivetarget for application of the inventive exon shuffling techniques inthat the co-linearity of functional domains and catalytic capabilitiesis so well established for this family.

Also, the close mechanistic relationship between class I polyketidesynthases and animal fatty acid synthases, class II polyketidesynthases, and/or intermediate class polyketide synthases (e.g., fungalpolyketide synthases, whose funcitonal organization and catalyticcharacteristics are apparently intermediate between those of thebacterial class I and class II polyketide synthases) renders shufflingreactions that admix DNAs encoding functional domains of two or more ofthese different families particularly intriguing. Such reactions willgenerate libraries of new synthetic enzymes, which in turn will generatelibraries of new chemical compounds that can be assayed according to anyavailable technique to determine whether they have interesting ordesirable biological activities.

Integration with Existing Technologies

It will be appreciated that the present invention does not describe theonly available method for linking selected nucleic acid molecules to oneanother. For example, the established restriction-enzyme-basedtechnology clearly allows cleavage and ligation of nucleic acidmolecules, albeit without the convenience and other advantages of theinventive system. Also, techniques have been developed by whichribozymes can be employed to mediate cleavage and ligation of nucleicacids at the RNA or DNA level (see, for example, U.S. Pat. No.5,498,531; U.S. Pat. No. 5,780,272; WO 9507351; WO 9840519, and U.S.Patent Application Ser. No. 60/101,328, filed Sep. 21, 1998, each ofwhich is incorporated herein by reference; see also Example 4).

Each of these different systems for nucleic acid manipulation offerscertain advantages and disadvantages. For example, ribozyme-mediatedsystems offer the distinct advantage that shuffling reactions may beperformed in vivo if desired (see, for example, U.S. Patent ApplicationSer. No. 60/101,328, filed Sep. 21, 1998). Furthermore, once a shufflingcassette is generated in which an exon of interest is linked to a firsttrans-splicing ribozyme component, that exon may be ligated to any otherexon that is linked to a second trans-splicing component that iscompatible with the first trans-splicing component in a simpletrans-splicing reaction. Thus, the more the ribozyme-mediated system isutilized, and the larger the number of shuffling cassettes generated byits use, the more powerful it becomes.

Ribozyme-mediated nucleic acid manipulation, like the techniquesdescribed herein, can be used for exon shuffling, and can be engineeredto direct seamless ligation of any selected nucleic acid molecules.Furthermore, like the inventive system, the ribozyme-mediated system maybe engineered so that the agents that mediate ligation (the ribozymecomponents in the ribozyme-mediated system; the overhangs in theinventive system) are only compatible with certain selected otherligation-mediating agents. This ability allows one to perform directedligation reactions analogous to those depicted in FIG. 17, in which acollection of exons is incubated together but only certain selectedexons can become ligated to one another (see, for example, Example 4 andFIG. 29).

One particularly preferred embodiment of the present inventionrepresents an integration of the primer-based manipulation techniquesdescribed herein with the ribozyme-mediated techniques described in theabove-referenced patents and patent applications. Specifically, theprimer-based nucleic acid manipulation techniques described herein areutilized to construct ribozyme-associated shuffling cassettes that arethen employed in splicing reactions to generate hybrid nucleic acidmolecules that can subsequently be cloned and manipulated usinginventive primer-based strategies.

FIG. 26 presents one version of such a combinedprimer-based/ribozyme-mediated nucleic acid manipulation scheme. Asdepicted, nine different product molecules are produced using inventiveprimer-based nucleic acid manipulation strategies. These molecules aredesigned to be ligated together to produce three different shufflingcassettes. The first shuffling cassette comprises (i) a promoter thatwill direct transcription of the cassette; (ii) a first tag sequence;(iii) an upstream terminal exon; and (iv) a first ribozyme component.The second shuffling cassette comprises (i) a promoter that will directtranscription of the cassette; (ii) a second ribozyme component,compatible with the first ribozyme component; (iii) an internal exon;and (iv) a third ribozyme component (optionally not compatible with thesecond ribozyme component). The third shuffling cassette comprises (i) apromoter that will direct transcription of the cassette; (ii) a fourthribozyme component that is compatible with the third ribozyme component(and optionally not with the first ribozyme component); (iii) adownstream terminal exon; and (iv) a second tag sequence.

Given the ease with which shuffling cassettes may be generated using theinventive primer-based technology, there is no need for shufflingcassettes to be introduced into vectors; they may be transcribeddirectly. Of course, they may be introduced into vectors if so desired,preferably by means of the inventive primer-based nucleic acidmanipulation techniques. Each cassette is transcribed and thetranscription products are incubated with one another under splicingconditions, either in vitro or in vivo, to produce a hybrid moleculecontaining each of the three exons. The hybrid molecule may then beintroduced into a vector or further manipulated, again preferably usingthe inventive primer-based manipulation technology.

Those of ordinary skill in the art will appreciate that more than oneinternal cassette may be employed in the system of FIG. 26, either in anexon shuffling (involving positional and/or identity shuffling) reactionor in a directed ligation reaction in which only one copy of each exonwill be introduced into the hybrid molecule, in a pre-determined order.Alternatively or additionally, multiple alternative upstream ordownstream exons may be employed, or such terminal exons may be leftout. In a particularly preferred embodiment of an identity exonshuffling reaction, multiple alternative exons are provided, and aresimultaneously shuffled, for at least two positions (e.g., one internalposition and one terminal position, two internal positions, or twoterminal positions) in the hybrid molecule.

One advantage of the combined primer-based/ribozyme-mediated systemdepicted in FIG. 26 can be appreciated through consideration of thenumber of primers required to generate the indicated molecules, and/orto clone them into vectors or other desirable locales, according to theinventive methods. For example, sixty-seven primers are required togenerate the initial product molecules if 10 different possible exonproduct molecules are produced for each of the “A”, “B”, and “C” exons.This is a relatively large number of primers, but is justified by theease with which the product molecules are generated and ligated togetherusing the inventive system, as compared with alternative methods (e.g.,standard restriction-enzyme-based cloning techniques) available for theproduction of the shuffling cassettes. Only four primers are required toamplify the resulting shuffling cassettes, or to ligate them to otherDNA molecules (e.g., a vector). Most importantly, only two primers arerequired to amplify (or ligate) assembled genes. Particularly where exonshuffling reactions have been performed, and a library of assembledgenes is generated, it is valuable to be able to amplify all members ofthe library with the same two primers.

Automation

One particularly attractive feature of the inventive techniques andreagents is their susceptibility to automation. In particular, wherelarge libraries of novel hybrid nucleic acids are being produced ininventive exon shuffling reactions, it may be desirable to employ anautomated system (e.g., the Beckman 2000 Laboratory Automation WorkStation) to accomplish the simultaneous manipulation of a large numberof different samples.

To give but one example of a preferred automated application of thepresent inventive methods, FIG. 27 depicts a robotic system that couldbe utilized, for example, to accomplish exon shuffling as depicted inFIG. 27 and further to screen the products of the shuffling reaction fordesired activities. For example, the product molecules depicted in thefirst column of FIG. 26 could be generated by PCR in 96 well platesusing a Biomek 2000 system in combination with a multimek 96 automated96-channel pipetter and a PTC-225 DNA engine (MJ Research), relying onthe ORCA robot arm to move the plates from one location to another asnecessary.

Preferably, multiple alternatives are simultaneously prepared of eachexon product molecule (e.g., n “A” exons, A1-An, are prepared; as are x“B” exons, B1-Bx; and y “C” exons, C1-Cy), along with T7/X, 1-4′,T7/5,6, and Y products. As discussed above, 67 different primers arerequired to produce these product molecules according to the inventivemethodologies described herein.

The automated system is then programmed to pipette the appropriateproduct molecules together, along with desired ligation reagents, toproduce 30 shuffling cassettes of the types depicted in the secondcolumn of FIG. 26. The system is then programmed to generate RNA fromthese shuffling cassettes using T7 RNA polymerase. The “A”-type,“B”-type, and “C”-type transcripts are then mixed together in allpossible combinations, and are incubated (still in the robotic system)under trans-splicing conditions. All together, 1000 different splicingreactions will be performed.

A small aliquot of each splicing reaction is then removed and amplifiedwith inventive primers so that the amplification products can readily beligated with a recipient molecule such as a vector. The resultingplasmids may then be introduced into host cells (e.g., bacterial cells)for further amplification, or alternatively may be introduced into an invivo or in vitro expression system so that any protein products encodedby the assembled shuffled genes may be assayed. Desirable expressionsystems will depend on the nature of the nucleic acid sequences thatwere shuffled. To give but one example, if fungal polyketide synthasegene fragments (e.g., encoding functional domains of fungal polyketidesynthase proteins) were shuffled according to this approach, it may bedesirable to express the hybrid proteins thereby generated in one ormore fungal or mammalian cells types in order to assess their syntheticcapabilities.

Kits

Reagents useful for the practice of the present invention may desirablybe provided together, assembled in a kit. Certain preferred kits willinclude reagents useful for both primer-mediated and ribozyme-mediatednucleic acid manipulation reactions.

EXAMPLES Example 1 Preparation and Ligation of Product Molecules with 5′Overhang Sequences

This Example describes the preparation and ligation of product moleculeshaving 5′ overhangs, using hybrid primers containingdeoxyribonucleotides at their 3′ ends and ribonucleotides at their 5′ends.

FIG. 18 presents a schematic of the particular experiment that wasperformed. As shown, three different product molecules were generated,two of which correspond to exons of the gene for subunit B of the humanglutamate receptor, and one of which corresponds to an intron from theunrelated human β-globin gene. The particular glutamate receptor exonswe utilized are known as Flip and Flop, and are indicated in FIGS. 19Aand 19B, which present the nucleotide sequences of each of these exons(GenBank accession numbers X64829 and X64830, respectively).

We prepared each of our three product molecules by PCR, using Vent® DNApolymerase and plasmids Human GluR-B #7 (a cloned genomic fragmentcontaining exons 13-16 of the human glutamate receptor B subunit) orHβT7 (a cloned genomic fragment containing exons 1-2 of the humanβ-globin gene).

The Flop exon was amplified with a 5′ primer (primer 1 in FIG. 18;5′-AAATGCGGTTAACCTCGCAG, SEQ ID NO 1) that is entirely DNA andcorresponds to the first 20 bases of the Flop exon, in combination witha 3′ primer (primer 2 in FIG. 18; 5′-accuTGGAATCACCTCCCCC SEQ ID NO 2)whose 5′-most four residues are RNA, as indicated by lower case lettersin FIG. 18. This primer corresponds to the last 18 bases of the Flopexon plus 2 bases of intron. Together, these primers amplify a fragmentcorresponding to all of the human glutamate receptor Flop exon (115basepairs) plus the first two residues at the 5′ end of the intron.

Intron 1 was amplified with a 5′ primer (primer 3 in FIG. 18;5′-agguTGGTATCAAGGTTACA, SEQ ID NO 3) whose sequence corresponds to thefirst 18 bases of the human β-globin intron 1, and whose 5′-most fourresidues are RNA, and are complementary to the four RNA residues at the5′ end of primer 2; in combination with a 3′ primer (primer 4 in FIG.18, 5′-cuAAGGGTGGGAAAATAGAC, SEQ ID NO 5) corresponding to the last 20bases of the human β-globin intron 1, whose 5′-most two residues areRNA. These primers together amplify a fragment corresponding to theentire intron (129 bp), and 2 add two residues corresponding to the lasttwo residues at the 3′ end of the Flop exon.

The Flip exon was amplified with a 5′ primer (primer 5 in FIG. 18,5′-agAACCCCAGTAAATCTTGC, SEQ ID NO 4) corresponding to the first 18bases of the human glutamate receptor Flip exon, whose 5′-most tworesidues are RNA and are complementary to the two RNA residues at the5′-end of primer 4; in combination with a 3′ primer (primer 6 in FIG.18, 5′-CTTACTTCCCGAGTCCTTGG, SEQ ID NO 6) corresponding to the last 20exon bases, that was entirely DNA. These primers together amplify afragment corresponding to the entire Flip exon (115 bp) and the last twonucleotides at the 3′ end of the intron.

Each amplification reaction included 400 μmole of each primer, kinased(using T4 polynucleotide kinase in 100 μl 1×NEB T4 ligase buffer [50 mMTris-HCl pH 7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA] for 30minutes at 37° C., followed by dilution to 10 pmol/μl with 200 μlnuclease-free dH₂O); 2 units Vent_(R)® (exo⁻) polymerase (NEB, Beverly,Mass.), 100 μl 1× Vent buffer (10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris, 2mM MgSO₄, 0.1% Triton X-100); 200 μM dNTPs; and 5 ng of templateplasmid. One cycle of (i) 95° C., 3 minutes; (ii) 60° C., 3 minutes;(iii) 72° C., 3 minutes was followed by 35 cycles of (i) 95° C., 15seconds; (ii) 60° C., 15 seconds; (iii) 72° C., 30 seconds, in aRobocycler® gradient 40 (Stratagene, La Jolla, Calif.) thermalcycler.

We found that Vent_(R)® and Vent_(R)® (exo-) did not copy theribonucleotides in our primers, so that, after amplification, eachproduct molecule contained a 5′ ribonucleotide overhang at one or bothends (4 nucleotides at the 3′-end of the Flop product; 4 nucleotides atthe 5′-end of the β-globin intron product; 2 nucleotides at the 3′-endof the β-globin intron product; and 2 nucleotides at the 5′-end of theFlip product).

Each amplified product was precipitated with ethanol (EtOH) and wasresuspended in 10 μL, 2 of which were run on a 6% polyacrylamide gel inorder to verify the presence of all three amplification products.Aliquots (2-4 μL each) containing approximately equimolar quantities ofeach fragment were then combined in a ligation reaction containing 1×New England Biolabs (NEB) T4 ligase buffer (50 mM Tris, pH 7.8, 10 mMMgCl₂, 10 mM DTT, 1 mM ATP, 25 μg/ml BSA) and 0.5 U of T4 DNA ligase(NEB, Beverly, Mass.). The 20 μL reaction was incubated overnight at 4°C. to allow ligation to occur. Products of ligation were then amplifiedusing primers 1 and 3 and Taq polymerase, which does copy RNA (Myers etal., Biochem. 6:7661, 1991). The amplification reaction contained 1× Taqbuffer (20 mM Tris, pH 9.0, 50 mM KCl, 0.1% Triton X-100), 200 μM dNTPs,5 Units of Taq polymerase (Promega, Madison, Wis.), 2 μL of the ligationmix, and 400 μmol of each primer.

The product of the Taq amplification is shown in FIG. 20, and wasligated into the PCR 2.1 vector (Invitrogen, Carlsbad, Calif.) using theTA Cloning Kit according to manufacturer's instructions. Sequenceanalysis (using standard dideoxy sequencing methods, and Universal andReverse primers from United States Biochemical, Cleveland, Ohio) ofmultiple (9) clones confirmed that all ligation junctions were correct(see FIGS. 21 and 22).

Because this strategy ligated product molecules with rubonucleotideoverhangs, it is sometimes referred to as Ribonucleotide overhangcloning (ROC).

Example 2 Preparation and Ligation of Product Molecules with 3′ OverhangSequences

This Example describes the preparation and ligation of product moleculeshaving 3′ overhangs, using hybrid primers containingdeoxyribonucleotides at their 3′ ends and ribonucleotides at their 5′ends.

FIG. 28 presents a schematic of the particular experiment that wasperformed. As shown, three different product molecules were generated,two of which correspond to the Flip and Flop exons of the gene forsubunit B of the human glutamate receptor, and one of which correspondsto an intron from the unrelated human β-globin gene (see Example 1).

Each of the three product molecules was prepared by PCR, using a Pfupolymerase which copies RNA nucleotides, and either human genomic DNA orHBT7 (see Example 1). The Flop exon was amplified with primers 1 and 2from Example 1; intron 1 was amplified either with primers 3 and 4 fromExample 1 or with primer 3 and an alternative primer 4(5′uucuAAGGGTGGGAAAATAG-3′; SEQ ID NO 24); the Flip exon was amplifiedeither with primers 5 and 6 or with an alternative primer 5(5′agaaCCCAGTAAATCTTGC; SEQ ID NO 25); and primer 6.

Each 100 μL reaction contained 2.5 U of Pfu Turbo® polymerase(Stratagene), 1× Cloned Pfu buffer (10 mM (NH₄)₂SO₄, 20 mM Tris pH=8.8,2 mM Mg SO₄, 10 mM KCl, 0.1% Triton X-100 and 0.1 mg/ml BSA), 200 μM ofeach dNTP, 1 mM MgSO₄, and primers at a final concentration of 0.5 μMeach. The Flop and Flip reactions contained 375 ng of human genomic DNA,while the β-globin reaction contained 5 ng of HBT7 DNA. The PCR stepprogram was one cycle of 95° C., 5 min; 50° C., 3 min; 72° C. 3 min;followed by 40 cycles of 95° C., 30 sec; 50° C., 30 sec; 72° C., 45 sec;followed by one cycle of 72° C., 5 min in Robocycler gradient 40 for theFlip and Flop fragments. The same program was used to amplify β-globinintron 1, except the annealing temperature was 46° C. Since Pfupolymerase does not copy RNA (stratagene product literature), the PCRproduct literature), the PCR products contained 5′ overhangs. Theseoverhangs were filled in during an incubatioin at 72° C. for 30 minuteswith 5 U of Tth polymerase (Epicentre Technolgies, Madison, Wis.), tofill in the 5′-RNA overhangs (Note, in more recent experiments, M-MLV RTwas used, rather than Tth, to fill in the overhangs. When M-MLV RT wasused, the fragments were separated on agarose gels prior to treatmentwith 200 U of M-MLV RT in 1× First strand buffer (50 mM Tris pH=8.3, 75mM KC1, 3 mM MgCl₂), 10 mM DTT and 0.5 mM dNTP in 20 μL.). This strategyallowed us to use Pfu polymerase, which has the highest fidelity ofavailable thermostable DNA polymerases, during the amplificationreaction but still generate blunt-ended reaction products.

The amplified parental PCR products were excised from an agarose gel andpurified. Five μl of each purified sample were fractionated on anagarose gel for quantitation. We then converted the blunt-ended productsto products containing 3′ overhangs by removing the ribonucleotidesthrough exposure to mild base. NaOH (1 N) was added to 8 μl of each ofthe gel isolated fragments to a final concentration of 0.2 N and thesamples were incubated at 45° C. for 30 min. The base was neutralized byaddition of 2 μl of 1 N HC1. Since NaOH hydrolysis generates a3′-phosphate and a 5′-OH, we had to phosphoylate the products to be ableto ligate them. The DNA fragments were phosphorylated in 1× T4 ligasebuffer (USB) in a total of 20 μl for 30 min at 37° C. using 10 U of PNK(USB). Approximately 25 ng (3-6 μl) of each phosphorylated product werecombined in a final volume of 20 μl and ligated for 16 hours at 14 μC in1× T4 ligase buffer with 5 Weiss U of T4 DNA ligase (USB).

To produce the chimeric Flop-β-Flip product, a secondary PCR amplicationwas performed as described above for the primary PCRs using 1 μl ofligation reaction as template, primers 1 and 6, and an annealingtemperature of 58° C. A chimeric product of the expected size (360 bp)was observed. This product was cloned and sequenced; both ligationjunctions were correct in 6 of 8 clones that were sequenced. Two cloneseach had an error at one of the ligation sites. In one clone, three basepairs were lost at the boundary between the β-globin intron and Flip. Inthe other clone, an A was changed to a T (data not shown). We suspectthat Tth polymerase introduced these errors during the fill in step ofthe procedure. Because the strategy described here involved ligation ofmolecules containing DNA overhangs, it is sometimes referred to as DNAOverhang Cloning (DOC).

Example 3 Bioassays for Determining Success of Primer Copying and/orLigation

The present Example describes techniques that could be used to evaluatethe ability of a particular DNA polymerase to copy (i.e., to use as atemplate) a particular modified oligonucleotide primer. For example, thetechniques described herein might be useful to determine whether aparticular modified nucleotide or ribonucleotide (or collection thereof)can be replicated by one or more DNA polymerases.

FIG. 29 presents one embodiment of the present bioassay techniques. Asshown, two primers are provided that hybridize with a template molecule.The first primer is known to be extendible by a particular DNApolymerase; the second primer includes one or more modified nucleotidesor ribonucleotides whose ability to block replication by the DNApolymerase is unknown. Any nucleotide modification may be studied in thesystem.

As shown in FIG. 29, both primers are extended, so that, if replicationis blocked, a product molecule with a 5′ overhang is produced; ablunt-ended product molecule (or a molecule containing asingle-nucleotide 3′-overhang, depending on the DNA polymerase employed)is generated if replication is not blocked.

The product molecule is then incubated with a vector containing acomplementary 5′ overhang and carrying a selectable marker (or a markeridentifiable by screening). Only if replication was blocked willhybridization occur. Ligation is then attempted and should succeedunless the particular modification interferes with ligation of a nick onthe complementary strand (unlikely) or the modification is present atthe 5′ end of the overhang and is of a character that interferes withligation to an adjacent 3′ end. In order to simplify the experiment andminimize the number of variables in any particular reaction, it isexpected that modifications will only be incorporated at the very 5′ endof a primer if their ability to block replication is already known andthe desire is to asses only their ability to interfere with ligation.

The ligation product is then introduced into host cells, preferablybacteria. Selectable (or otherwise identifiable) cells will grow andproliferate only if the modification in question did block replicationand either (i) did not block ligation on the complementary strand; or(ii) did block ligation on the complementary strand but did not block invivo nick repair. If the modification were at the 5′ end of the primer,cells will only grow if the modification did block replication and didnot block ligation of both strands.

Of course, where the modification constitutes one or moreribonucleotides, or other removable nucleotides, absence of colonies dueto inability to block replication can be distinguished from otherabsence of colony results by treating the original product molecule withan agent that will remove the modified nucleotide(s), along with anymore 5′ nucleotides, and then incubating the resulting secondary productmolecule, which contains a 3′ overhang complementary to the modifiednucleotide and any more 5′ nucleotides, with a vector containing acompatible 3′ overhang.

Example 4 Directional Ligation of Multiple Nucleic Acid Molecules byEngineered Selective Compatibility of Catalytic Ribozyme Elements

FIG. 30 shows a directional ligation reaction that allowed selectiveligation of particular exons through use of incompatible ribozymecomponents. As indicated, transcripts were generated in which (i) afirst exon (A) was linked to a first ribozyme component from the aI5γgroup II intron; (ii) a second exon (B) was flanked by (a) a secondribozyme component, also from the aI5γ group II intron, that iscompatible with the first ribozyme component, and (b) a third ribozymecomponent, from the LTRB intron of Lactococus lacti, that is notcompatible with the second intron component; and (iii) a third exon waslinked to a fourth ribozyme component, also from the LTRB intron, thatis compatible with the third intron component but not with the firstintron component. These three transcripts were incubated together undersplicing conditions and, as shown, only the ABC product (and not the ACnor the circular B product) was produced.

In all, nine plasmids were used in the study: pJD20, pB.E5.D4,pD4.E3(dC).B(2), pLE12, pB.5′Lac, p3′Lac.B, pD4.E3(dC)B(2).5′Lac, andp3′Lac.B.E5.D4. Two PCR amplifications were performed using plasmidpJD20, which contains the full-length aI5γ intron (Jarrell et al., Mol.Cell. Biol. 8:2361, 1988), as a template. The first reaction amplifiedpart of the intron (domains 1-3 and 73 nt of domain 4), along with part(27 nt) of the 5′ exon. The primers utilized, BamHI.E5(5′-ACGGGATCCATACTTACTACGTGGTGGGAC; SEQ ID NO 7) and D4.SalI(5′-ACGGTCGACCCTCCTATCTTTTTTAATTTTTTTTT; SEQ ID NO 8), were designed sothat the PCR product had unique BamHI and SalI sites at its ends. ThePCR product was digested with BamHI and SalI, and was ligated into thePBS-vector (Stratagene), digested with the same enzymes, so that it waspositioned downstream of the T7 promoter. The resulting plasmid wasdesignated pB.E5.D4, and encodes the B.5′γ shuffling cassette (see FIG.31).

The second PCR reaction that utilized pJD20 as a template amplified adifferent part of the intron (the remaining 65 nt of domain 4 plusdomains 5-6), along with part (29 nt) of the 3′ exon. The primersutilized, KpnI.D4 (5′-ACGGGTACCTTTATATATAACTGATAAATATTTATT; SEQ ID NO 9)and E3.BamHI (5′-ACGGGATCCAGAAAATAGCACCCATTGATAA; SEQ ID NO 10), weredesigned so that the PCR product had unique KpnI and BamHI sites at itsends. The PCR product was digested with KpnI and BamHI, and was ligatedinto the PBS-vector, digested with the same enzymes, so that it waspositioned downstream of the T7 promoter. The resulting plasmid wascalled pD4.E3(dC).B (see FIG. 31).

Sequence analysis of the pD4.E3(dC).B plasmid revealed an unexpectedpoint mutation in the 3′ exon sequence. The expected sequence wasACTATGTATTATCAATGGGTGCTATTTTCT (SEQ ID NO 11); the observed sequence wasACTATGTATTATAATGGGTGCTATTTTCT (SEQ ID NO 12).

A site directed mutagenesis reaction was then performed, using theQuickChange® Site-Directed Mutagenesis Kit (Stratagene, catalog number200518) to insert an additional BamHI site into the 3′ exon sequence.The primers utilized were designated E3.BamHI(2)(5′-CTCTAGAGGATCCAGAAAATAGGATCCATTATAATACATAGTATCCCG; SEQ ID NO 13) andE3.BamHI(2)complement(5′-CGGGATACTATGTATTATAATGGATCCTATTTTCTGGATCCTCTAGAG; SEQ ID NO 14). Theplasmid generated as a result of the site-directed mutagenesis reactionwas designated pD4.E3.(dC).B(2), and encoded the 3′γ.B shufflingcassette (see FIG. 31), in which the length of the 3′ exon was shortenedto 13 nt.

Two additional PCR reactions were performed, in which the plasmid pLE12, which encodes the full-length LTRB intron flanked by its natural 5′and 3′ exons (Mills et al., J. Bacteriol. 178:3531, 1996), was used as atemplate. In the first reaction, primers 5′transM.E.5′(5′-CACGGGATCCGAACACATCCATAACGTGC; SEQ ID NO 15) and 5′sht3′(5′-CAGCGTCGACGTACCCCTTTGCCATGT; SEQ ID NO 16) were used to amplify partof the LTRB intron (domains 1-3), and part (15 nt) of the 5′ exon. ThePCR product was generated with Taq polymerase and was cloned into thePCR2.1 Topo vector (Invitrogen) using the Topo® TA Cloning® kit(Invitrogen). The resulting plasmid was designated pB.5′Lac, and encodesthe B.5′Lac shuffling cassette (see FIG. 31).

The same PCR product was also digested with BamHI and SalI, and wasligated into pD4.E3(dC).B(2), cut with the same enzymes, to producepD4.E3(dC)B(2).5′Lac, which encodes the 3′γ.B.5′Lac shuffling cassette(see FIG. 31).

Additionally, plasmid pB.5′Lac was digested with SpeI and Asp718 toremove some unwanted restriction sites. Overhangs were filled in withKlenow fragment, and the resulting blunt ends were ligated to reseal thevector. The plasmid thereby produced was designated pB.5′Lac(K) (seeFIG. 31).

The second PCR reaction that utilized pLE12 as a template involved theuse of primers 3′transM.E.5′(5′-CACGGAGCTCTTATTGTGTACTAAAATTAAAAATTGATTAGGG; SEQ ID NO 17) and3′transM.E.3′ (5′-CAGCGGATCCCGTAGAATTAAAAATGATATGGTGAAGTAG; SEQ ID NO18) to amplify part of the PTRB intron (domains 4-6), attached to part(21 nt) of the 3′ exon. The primers were designed so that the PCRproduct had unique SacI and BamHI sites at its ends. The PCR productswas generated with Taq polymerase and was cloned into the pCR2.1 Topovector. The resulting plasmid was designated 3′Lac.B, and encoded the3′Lac.B shuffling cassette (see FIG. 31).

Plasmid p3′Lac.B was digested with SacI and BamHI, and the 1993 bp bandthereby generated was purified from an agarose gel using the GenecleanII kit (BIO 101). The purified fragment was then ligated into pE5.D4,digested with the same enzymes, to produce plasmid p3′Lac.B.E5.D4,encoding the 3′Lac.B.5′γ shuffling cassette (see FIG. 31).

Plasmids pB.E5.D4, pD4.E3(dC).B(2), pB.5′Lac, p3′Lac,B, andpD4.E3(dC)B(2).5′Lac were linearized with HindIII and were transcribedin vitro with T7 RNA polymerase (Stratagene, catalog number 600123) at40° C. for 1 hour in 100 μL reactions containing 6 μg of linearizedtemplate DNA and 0.5 mM unlabeled ATP, CTP, GTP, and UTP. The RNAsproduced in these transcription reactions were treated with 1 U of RQ1RNase-free DNase, were extracted with phenol-chloroform, were desaltedon a Sephadex G25 column, and were precipitated with EtOH. Precipitateswere subsequently resuspended in 6 μL water.

One μL of each resuspended RNA transcript was then used in atrans-splicing reaction carried out at 45° C. for 60 minutes, in 40 mMTris-HCl, pH 7.6, 100 mM MgCl₂, and either 0.5 M NH₄Cl or 0.5M(NH₄)₂SO₄.

After the trans-splicing reaction, a reverse transcription/PCR reactionwas performed to identify ligated splicing products. The detectedproducts were: (i) ligated alγ5 exons E5 and E3 produced bytrans-splicing of B.E5.D4 and D4.E3(dC).B(2) (lane 1, FIG. 32); (ii)ligated LTRB 5′ and 3′ exons produced by trans-splicing of 3′Lac.B and3′Lac.B (lane 2, FIG. 33); and (iii) the three-molecule ligation productproduced by trans-splicing of B.E5.D4, D4.E3(dC).B(2).5′Lac, and 3′Lac.B(lanes 2 and 3, FIG. 33).

Example 5 Cloning Products of 3′-Overhang Product Ligation withoutAmplification of Chimeric Product

We found that the products of a DOC ligation reaction could be cloneddirectly into a vector for replication in bacteria without a chimericamplification step. As was described above in Example 2, we designedchimeric primers that, when used in a DOC experiment, generated Flop,intron 1, and Flip PCR products that could be ligated directionally. Inaddition, the primers were designed such that NaOH treatment of the PCRproducts creates an upstream overhang on the Flop exon that iscompatible with an Apa I overhang, and a downstream overhang on the Flipexon that is compatible with a Pst I overhang. All three fragments wereincubated together in the presence of ligase and pBluescript II SK (−)that had been digested with ApaI and PstIl. An aliquot of the ligationmixture was transformed directly into E. coli, and the expected chimericclone was readily isolated, sequenced, and found to be perfect (data notshown).

Example 7 Construction of Multiple Chimeric Products by DNA-OverhangCloning

TO demonstrate the generality of the procedures described herein, weapplied the techniques of Example 2 and to a variety of differentmolecules and produced five different chimeras, shown in FIG. 35. Allfive chimeras were generated by directional three-molecule ligation.Note that these chimeras were generated using M-MLV reversetranscriptase, rather than Tth, to fill in 5′ RNA overhangs. When M-MLVRT was used, no errors were detected at any of the ligation points.

Other Embodiments

Those of ordinary skill in the art will appreciate that the foregoinghas been a description merely of certain preferred embodiments of thepresent invention; this description is not intended to limit the scopeof the invention, which is defined with reference to the followingclaims:

1. A method of generating a recombinant double-stranded DNA molecule,the method comprising the steps of: (a) providing a template nucleicacid molecule; (b) contacting the template nucleic acid molecule with afirst primer that hybridizes thereto, the first primer including atleast one termination residue characterized in that the terminationresidue serves as a replication template for a first DNA polymerase, butdoes not serve as a replication template for a second DNA polymerasethat is different from the first; (c) extending the first primer togenerate a complementary nucleic acid strand; (d) contacting thecomplementary nucleic acid strand with a second primer that hybridizesthereto; (e) extending the second primer with said second DNApolymerase, so that the termination residue is not copied in theextension reaction and the extension reaction produces a firstdouble-stranded DNA molecule containing a first 5′ overhang; (f)providing a second double-stranded DNA molecule containing a secondoverhang at least partly complementary with the first overhang; (g)forming an annealed DNA molecule by combining the first and seconddouble-stranded DNA molecules under conditions that allow hybridizationof the first and second overhangs; and (h) generating the recombinantdouble-stranded DNA molecule by exposing the annealed double-strandedDNA molecule to said first polymerase so that the termination residue iscopied into said recombinant double-stranded DNA molecule.
 2. The methodof claim 1 wherein steps (a)-(e) are repeated, using the double-strandedDNA molecule produced in step (e) as the template, such that, throughiteration, multiple copies of the first double-stranded DNA molecule areproduced.
 3. The method of claim 1, wherein step (h) comprises exposingthe annealed double-stranded DNA molecule to said first polymerase invitro.
 4. The method of claim 1, wherein step (h) comprises exposing theannealed double-stranded DNA molecule to said first polymerase in vivo.5. The method of claim 4, wherein the annealed double-stranded DNAmolecule is a nicked annealed double-stranded DNA molecule exposed invivo to said first polymerase without prior ligation.
 6. The method ofclaim 1, wherein the second primer includes at least one secondtermination residue characterized in that the second termination residueserves as a replication template for said first DNA polymerase, but doesnot serve as a replication template for said second DNA polymerase, suchthat the first double-stranded DNA molecule exhibits two 5′ overhangs.7. The method of claim 6, wherein the termination residue and the secondtermination residue are the same.
 8. The method of claim 6, wherein thetwo 5′ overhangs have identical sequences.
 9. The method of claim 6,wherein the two 5′ overhangs have complementary sequences.
 10. Themethod of claim 6, wherein the two 5′ overhangs have differentsequences.
 11. The method of claim 1 or claim 6, wherein the step (a)comprises providing a plurality of different template nucleic acidmolecules.
 12. The method of claim 11, wherein each of the differenttemplate nucleic acid molecules hybridizes with the same first primer.13. The method of claim 11, wherein the step (b) comprises contactingthe plurality of different template nucleic acid molecules with aplurality of different first primers, such that different primershybridize with different template nucleic acid molecules.
 14. The methodof claim 13, wherein each different template hybridizes with a differentfirst primer.
 15. The method of claim 12, wherein each complementarynucleic acid strand produced by extension of the first primer hybridizeswith the same second primer.
 16. The method of claim 12, wherein eachcomplementary nucleic acid strand produced by extension of the firstprimer does not hybridize with the same second primer and the step (d)comprises contacting the complementary nucleic acid strands with aplurality of different second primers so that different second primershybridize with different complementary nucleic acid strands.
 17. Themethod of claim 13, wherein each complementary nucleic acid strandproduced by extension of the plurality of different first primershybridizes with the same second primer.
 18. The method of claim 13,wherein each complementary nucleic acid strand produced by extension ofthe plurality of different first primers does not hybridize with thesame second primer and the step (d) comprises contacting thecomplementary nucleic acid strands with a plurality of different secondprimers so that different second primers hybridize with differentcomplementary nucleic acid strands.
 19. The method of claim 11, whereinreactions using different template molecules are performed in differentvessels.
 20. The method of claim 19, wherein each reaction using adifferent template molecule is performed in a different vessel.
 21. Themethod of claim 13, wherein reactions using different first primers areperformed in different vessels.
 22. The method of claim 21, wherein eachreaction using a different first primer is performed in a differentvessel.
 23. The method of claim 16, wherein reactions using differentsecond primers are performed in different vessels.
 24. The method ofclaim 23, wherein each reaction using a different second primer isperformed in a different vessel.
 25. The method of claim 18, whereinreactions using different second primers are performed in differentvessels.
 26. The method of claim 25 wherein each reaction using adifferent second primer is performed in a different vessel.
 27. Themethod of claim 1 or claim 6, further comprising: (a′) providing aplurality of reaction vessels; and (b′) performing steps (a)-(e)separately in each reaction vessel.
 28. The method of claim 27, whereineach reaction vessel comprises a well in a multi-well plate.
 29. Themethod of claim 1, wherein the step (f) comprises providing a pluralityof different second double-stranded DNA molecules.
 30. The method ofclaim 28, wherein the step (g) comprises forming a plurality ofdifferent annealed DNA molecules comprising different seconddouble-stranded DNA molecules.
 31. The method of claim 30 wherein steps(a)-(e) are performed with one or more pluralities selected from thegroup consisting of a plurality of different template nucleic acids, aplurality of different first primers, a plurality of different secondprimers, and combinations thereof, such that a plurality of differentfirst double-stranded DNA molecules is produced in step (e).
 32. Themethod of claim 31, wherein different first double stranded DNAmolecules are produced in different reaction vessels.
 33. The method ofclaim 30, wherein different annealed DNA molecules are generated indifferent reaction vessels.
 34. The method of claim 32, whereindifferent first double-stranded DNA molecules are hybridized withdifferent second double-stranded DNA molecules in different reactionvessels.
 35. The method of claim 1, wherein the first double-strandedDNA molecule comprises a first protein-coding sequence.
 36. The methodof claim 35, wherein the second double-stranded DNA molecule alsocomprises a second protein-coding sequence.
 37. The method of claim 36,wherein the step (h) of generating the recombinant double-stranded DNAmolecule comprises linking the first and second protein-coding sequencesto generate an in-frame fusion.
 38. The method of claim 27, wherein eachof the first double-stranded DNA molecules comprises a firstprotein-coding sequence.
 39. The method of claim 37 wherein each of thefirst double-stranded DNA molecule comprises a different firstprotein-coding sequence.
 40. The method of claim 38, wherein the seconddouble-stranded DNA molecule also comprises a second protein-codingsequence that is different from the first protein-coding sequence. 41.The method of claim 40, wherein step (h) comprises linking the first andsecond protein-coding sequences to generate an in-frame fusion.
 42. Themethod of claim 30, wherein each different first double-stranded DNAmolecule encodes a different protein-coding sequence.
 43. The method ofclaim 42 wherein the step (f) comprises providing a seconddouble-stranded DNA molecule that encodes a protein-coding sequence. 44.The method of claim 43, wherein the step (h) comprises generating aplurality of different recombinant DNA molecules in which differentfirst double-stranded DNA molecules are linked with the seconddouble-stranded DNA molecules to generate a plurality of in-framefusions.
 45. The method of claim 44, wherein different recombinant DNAmolecules are generated in a reaction vessel.
 46. The method of claim44, wherein different recombinant DNA molecules are generated indifferent reaction vessels.
 47. The method of claim 43, wherein the step(f) comprises providing a plurality of different second double-strandedDNA molecules encoding different protein-coding sequences.
 48. Themethod of claim 47, wherein the step (h) comprises generating aplurality of different recombinant DNA molecules in which differentfirst double stranded DNA molecules are linked with different seconddouble-stranded DNA molecules to generate a plurality of in-framefusions.
 49. The method of claim 48, wherein different recombinant DNAmolecules are generated in a reaction vessel.
 50. The method of claim48, wherein different recombinant DNA molecules are generated indifferent reaction vessels.
 51. A method of generating a product DNAmolecule, the method comprising steps of: (a) providing a template DNAmolecule containing a first target sequence; (b) contacting the templateDNA molecule with a first primer that hybridizes to said first targetsequence, the first primer including a ribonucleotide residue thatfunctions to block extension by a first DNA polymerase but does notblock extension by a second DNA polymerase; (c) extending the firstprimer to generate a complementary nucleic acid strand that contains asecond target sequence; (d) contacting the complementary nucleic acidstrand with a second primer that hybridizes to said second targetsequence; (e) extending the second primer with said first DNA polymerasewherein the ribonucleotide residue blocks extension by said firstpolymerase, producing a double stranded DNA molecule containing a first5′ overhang sequence; (f) combining the double stranded DNA moleculewith a recipient DNA molecule containing a second 5′ overhang sequenceat least partly complementary with the first 5′ overhang sequence toproduce an annealed DNA molecule; and (g) generating the product DNAmolecule by exposing the annealed DNA molecule to said second polymeraseso that the ribonucleotide residue is copied into said product DNAmolecule.
 52. A product DNA molecule produced according to a methodcomprising (a) amplifying a target nucleotide sequence with a firstpolymerase and a first and a second primer, wherein at least one of thefirst and second primers contains at least one terminator residue thatfunctions to block extension by said first polymerase, so that anamplicon is produced that contains at least a first 5′ overhangsequence; (b) combining said amplicon with a recipient moleculecontaining a second 5′ overhang sequence complementary to said first 5′overhang sequence to produce an annealed DNA molecule; and (c) exposingsaid annealed DNA molecule to a second polymerase that is different fromthe first polymerase, said second polymerase functioning to copy saidterminator residue into said product DNA molecule.
 53. A method forproducing a product DNA molecule encoding a desired protein sequencecomprising: (a) amplifying a target nucleotide sequence with a firstpolymerase and a first and a second primer, wherein at least one of thefirst and second primers contains at least one terminator residue thatfunctions to block replication by said first polymerase, so that anamplified product is produced that contains at least a first 5′ overhangsequence; (b) combining said amplified product with a recipient moleculecontaining a second 5′ overhang sequence complementary to said first 5′overhang sequence to produce a annealed DNA molecule; and (c) exposingsaid annealed DNA molecule to a second polymerase that is different fromthe first polymerase, said second polymerase functioning to copy saidribonucleotide residue into said product DNA molecule.
 54. A method forforming a double stranded DNA molecule comprising a 5′ overhangsequence, said method comprising the steps of: (a) providing a templatenucleic acid molecule; (b) contacting the template nucleic acid moleculewith a first primer that hybridizes thereto, the first primer includingat least one termination residue characterized in that the terminationresidue serves as a replication template for a first DNA polymerase, butdoes not serve as a replication template for a second DNA polymerasethat is different from the first; (c) extending the first primer togenerate a complementary nucleic acid strand; (d) contacting thecomplementary nucleic acid strand with a second primer that hybridizesthereto; (e) extending the second primer with said second DNApolymerase, so that the termination residue is not copied in theextension reaction and the extension reaction produces a firstdouble-stranded DNA molecule containing a first 5′ overhang sequence.55. A method for forming a double stranded DNA molecule comprising a 5′overhang sequence, said method comprising the steps of: (a) providing atemplate DNA molecule containing a first target sequence; (b) contactingthe template DNA molecule with a first primer that hybridizes to saidfirst target sequence, the first primer including a ribonucleotideresidue that functions to block extension by a first DNA polymerase butdoes not block extension by a second DNA polymerase; (c) extending thefirst primer to generate a complementary nucleic acid strand thatcontains a second target sequence; (d) contacting the complementarynucleic acid strand with a second primer that hybridizes to said secondtarget sequence; (e) extending the second primer with said first DNApolymerase wherein the ribonucleotide residue blocks extension by saidfirst polymerase, producing a first double stranded DNA moleculecontaining a first 5′ overhang sequence.
 56. A method for forming a 5′overhang sequence in a double stranded DNA molecule comprising: (a)providing a template DNA molecule containing a first target sequence;(b) contacting the template DNA molecule with a first primer thathybridizes to said first target sequence, the first primer including aribonucleotide residue that functions to block extension by a first DNApolymerase but does not block extension by a second DNA polymerase; (c)extending the first primer to generate a complementary nucleic acidstrand that contains a second target sequence; (d) contacting thecomplementary nucleic acid strand with a second primer that hybridizesto said second target sequence; (e) extending the second primer withsaid first DNA polymerase wherein the ribonucleotide residue blocksextension by said first polymerase, producing a first double strandedDNA molecule containing a 5′ overhang sequence.